Compositions and Methods for Lowering Triglycerides without Raising LDL-C Levels in a Subject on Concomitant Statin Therapy

ABSTRACT

In various embodiments, the present invention provides compositions and methods for treating and/or preventing cardiovascular-related diseases in subject in need thereof.

PRIORITY CLAIM

The present application is a continuation of U.S. patent application Ser. No. 16/197,924 filed Nov. 21, 2018, which is a continuation of U.S. patent application Ser. No. 14/441,111 filed on May 6, 2015, which is a 371 U.S. national stage application of PCT/US2013/068647 filed Nov. 6, 2013, which claims priority to U.S. Provisional Patent Application Nos. 61/723,108 filed on Nov. 6, 2012, 61/747,689 filed on Dec. 31, 2012, 61/761,398 filed on Feb. 6, 2013, and 61/830,463 filed on Jun. 3, 2013, the entire contents of each of which are incorporated herein by reference and relied upon.

BACKGROUND

Cardiovascular disease is one of the leading causes of death in the United States and most European countries. It is estimated that over 70 million people in the United States alone suffer from a cardiovascular disease or disorder including but not limited to high blood pressure, coronary heart disease, dyslipidemia, congestive heart failure and stroke.

SUMMARY

In various embodiments, the present invention provides pharmaceutical compositions and methods of using such compositions to treat and/or prevent cardiovascular-related diseases. In one embodiment, the subject is on concomitant statin therapy. In another embodiment, the subject on statin therapy has a baseline fasting serum triglyceride level of about 200 mg/dL to about 500 mg/dL.

In one embodiment, the invention provides a method of lowering triglycerides in a subject on stable statin therapy having baseline fasting triglycerides of about 200 mg/dl to about 500 mg/dl, the method comprising administering to the subject a pharmaceutical composition comprising polyunsaturated fatty acids, for example about 1 g to about 4 g of EPA per day, wherein upon administering the composition to the subject daily for a period of 12 weeks the subject exhibits at least 5% lower fasting triglycerides than a control subject maintained on stable statin therapy (optionally with placebo matching the EPA) without concomitant EPA for a period of 12 weeks wherein the control subject also has baseline fasting triglycerides of about 200 mg/dl to about 500 mg/dl. In another embodiment, upon administering the composition to the subject daily for a period of 12 weeks the subject exhibits no serum LDL-C increase, no statistically significant serum LDL-C increase, a serum LDL-C decrease, or the subject is statistically non-inferior to the control subjects (statin plus optional placebo) in regard to serum LDL-C elevation).

In various embodiments, the present invention provides pharmaceutical compositions and methods of using such compositions to increase plasma, serum and/or red blood cell (RBC) EPA levels and/or to treat or prevent cardiovascular-related diseases.

In one embodiment, a pharmaceutical composition according to the present disclosure comprises about 1 g of ethyl eicosapentaenoate and provides a mean plasma C_(max) of about 154.9+/−49.4 μg/mL, a mean plasma AUC_(0-24h) of about 2907+/−1160 μg·h/mL, a median plasma T_(max) of about 5 hours, and a mean plasma T_(1/2) of about 75.1+/−46.5 hours of ethyl eicosapentaenoate when administered orally twice per day to a human subject.

In another embodiment, a pharmaceutical composition comprises about 2 g of ethyl eicosapentaenoate and provides a mean plasma C_(max) of about 347.2+/−112.5 μg/mL, a mean plasma AUC_(0-24h) of about 6519+/−1963 μg·h/mL, a median plasma T_(max) of about 5 hours, and a mean plasma T_(1/2) of about 89.3+/−42.0 hours of ethyl eicosapentaenoate when administered orally twice per day to a human subject.

These and other embodiments of the present invention will be disclosed in further detail herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mean placebo-adjusted changes from baseline in plasma and RBC concentrations of EPA after 12 weeks of treatment with 2 g/day or 4 g/day of ultra-pure EPA in subjects on statin therapy and having baseline triglycerides of about 200 mg/dL to less than 500 mg/dL.

FIG. 2 depicts placebo-adjusted least square mean changes from baseline in plasma and RBC concentrations and ratios of selected fatty acids and fatty acid groups in subjects on statin therapy and having baseline triglycerides of about 200 mg/dL to less than 500 mg/dL.

FIG. 3 depicts the dose dependence of EPA concentrations in plasma and RBCs in subjects on statin therapy and having baseline triglycerides of about 200 mg/dL to less than 500 mg/dL.

FIG. 4A depicts the relationship between plasma triglyceride-lowering and EPA concentrations in plasma (a pharmacokinetic/pharmacodynamic relationship) in subjects on statin therapy and having baseline triglycerides of about 200 mg/dL to less than 500 mg/dL.

FIG. 4B depicts the relationship between plasma triglyceride-lowering and EPA concentrations in RBCs (a pharmacokinetic/pharmacodynamic relationship) in subjects on statin therapy and having baseline triglycerides of about 200 mg/dL to less than 500 mg/dL.

FIG. 5 depicts the mean placebo-adjusted changes from baseline in in plasma and red blood cell concentrations of EPA after 12 weeks of treatment with 2 g/day or 4 g/day of ultra-pure EPA in subjects having baseline triglycerides of at least about 500 mg/dL.

FIGS. 6A-6B depicts placebo-adjusted least square mean changed from baseline in plasma and red blood cell concentrations and ratios of selected fatty acids and fatty acid groups for 4 g/day (FIG. 6A) and 2 g/day (FIG. 6B) doses of ultra-pure EPA in subjects having baseline triglycerides of at least about 500 mg/dL.

FIG. 7 depicts the dose dependence of EPA concentrations in plasma and red blood cells in subjects having baseline triglycerides of at least about 500 mg/dL.

FIG. 8A depicts the relationship between plasma triglyceride-lowering and EPA concentrations in plasma (a pharmacokinetic/pharmacodynamic relationship) in subjects having baseline triglycerides of at least about 500 mg/dL.

FIG. 8B depicts the relationship between plasma triglyceride-lowering and EPA concentrations in red blood cells (a pharmacokinetic/pharmacodynamic relationship) in subjects having baseline triglycerides of at least about 500 mg/dL.

FIG. 9 depicts the distribution and disposition of subjects among four dosing groups, referred to herein as Groups 1 to 4.

FIG. 10 depicts the mean total plasma eicosapentaenoic acid concentration over time for dosing Groups 1 to 4. Bars indicate one standard deviation at each point.

FIG. 11 depicts the mean total plasma eicosapentaenoic acid trough concentration (C_(min)) over time for dosing Groups 1 to 4. Bars indicate one standard deviation at each point.

FIG. 12A depicts the mean total eicosapentaenoic acid plasma AUC_(0-24h) measured 28 days after onset of dosing for dosing Groups 1 to 4. FIG. 12B depicts the mean unesterified eicosapentaenoic acid plasma AUC_(0-24h) measured 28 days after onset of dosing for dosing Groups 1 to 4. FIG. 12C depicts the mean total eicosapentaenoic acid red blood cell (RBC) AUC_(0-24h) measured 28 days after onset of dosing for dosing Groups 1 to 4. Bars in FIGS. 12A to 12C indicate one standard deviation.

FIG. 13A depicts the mean total eicosapentaenoic acid plasma C_(max) measured 28 days after onset of dosing for dosing Groups 1 to 4. FIG. 13B depicts the mean unesterified eicosapentaenoic acid plasma C_(max) measured 28 days after onset of dosing for dosing Groups 1 to 4. FIG. 13C depicts the mean total eicosapentaenoic acid red blood cell (RBC) C_(max) measured 28 days after onset of dosing for dosing Groups 1 to 4. Bars in FIGS. 13A to 13C indicate one standard deviation.

FIG. 14 depicts median triglyceride percent change from baseline (y-axis) vs. mean plasma EPA concentration percent change from baseline (x-axis) for subjects administered placebo, EPA 2 g per day and EPA 4 grams per day. Bars in FIG. 14 indicate 95% confidence intervals for both axes.

FIG. 15 depicts mean trough total EPA plasma concentrations at baseline and end-of-treatment for various clinical studies.

FIG. 16 shows IPE Dose Dependence of RBC EPA Concentrations in the Phase 1 Pharmacokinetic (“PK”), MARINE, and ANCHOR studies.

FIG. 17 shows IPE Dose Dependence of Plasma EPA Concentrations in the Phase 1 PK, MARINE, and ANCHOR studies.

FIGS. 18A-18B show median percent changes from baseline for plasma triglycerides as a function of mean changes in EPA concentration in plasma (FIG. 18A) and red blood cells (FIG. 18B) for subjects in the MARINE study.

FIGS. 19A-19B show median percent changes from baseline for plasma triglycerides as a function of mean changes in EPA concentration in plasma (FIG. 19A) and red blood cells (FIG. 19B) for subjects in the ANCHOR study.

DETAILED DESCRIPTION

While the present invention is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the invention, and is not intended to limit the invention to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the invention in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values recited as well as any ranges that can be formed by such values. Also disclosed herein are any and all ratios (and ranges of any such ratios) that can be formed by dividing a disclosed numeric value into any other disclosed numeric value. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios can be unambiguously derived from the numerical values presented herein and in all instances such ratios, ranges, and ranges of ratios represent various embodiments of the present invention.

In one embodiment, the invention provides a method for treatment and/or prevention of cardiovascular-related diseases. The term “cardiovascular-related disease” herein refers to any disease or disorder of the heart or blood vessels (i.e. arteries and veins) or any symptom thereof. Non-limiting examples of cardiovascular-related disease and disorders include hypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia, coronary heart disease, vascular disease, stroke, atherosclerosis, arrhythmia, hypertension, myocardial infarction, and other cardiovascular events.

The term “treatment” in relation a given disease or disorder, includes, but is not limited to, inhibiting the disease or disorder, for example, arresting the development of the disease or disorder; relieving the disease or disorder, for example, causing regression of the disease or disorder; or relieving a condition caused by or resulting from the disease or disorder, for example, relieving, preventing or treating symptoms of the disease or disorder. The term “prevention” in relation to a given disease or disorder means: preventing the onset of disease development if none had occurred, preventing the disease or disorder from occurring in a subject that may be predisposed to the disorder or disease but has not yet been diagnosed as having the disorder or disease, and/or preventing further disease/disorder development if already present.

In one embodiment, the present invention provides a method of blood lipid therapy comprising administering to a subject or subject group in need thereof a pharmaceutical composition as described herein. In another embodiment, the subject or subject group has hypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia, borderline high/high triglycerides (e.g., about 200 to about 499 mg/dl), and/or very high triglycerides (e.g., at least about 500 mg/dl).

In another embodiment, the subject or subject group being treated has a baseline triglyceride level (or mean or median baseline triglyceride level in the case of a subject group), fed or fasting, of about 200 mg/dl to about 500 mg/dl. In another embodiment, the subject or subject group has a baseline LDL-C level (or mean or median baseline LDL-C level), despite stable statin therapy, of about 40 mg/dl to about 115 or about 40 to about 100 mg/dl. In another embodiment, the subject or subject group being treated has a baseline triglyceride level (or median baseline triglyceride level in the case of a subject group), fed or fasting, of at least about 200 mg/dl, at least about 300 mg/dl, at least about 400 mg/dl, at least about 500 mg/dl, at least about 600 mg/dl, at least about 700 mg/dl, at least about 800 mg/dl, at least about 900 mg/dl, at least about 1000 mg/dl, at least about 1100 mg/dl, at least about 1200 mg/dl, at least about 1300 mg/dl, at least about 1400 mg/dl, or at least about 1500 mg/dl, for example about 200 mg/dl to about 499 mg/dl, about 400 mg/dl to about 2500 mg/dl, about 450 mg/dl to about 2000 mg/dl or about 500 mg/dl to about 1500 mg/dl.

In another embodiment, the subject or subject group being treated has a baseline triglyceride level (or mean or median baseline triglyceride level in the case of a subject group), fed or fasting, of about 200 mg/dl to about 499 mg/dl. In another embodiment, the subject or subject group has a baseline LDL-C level (or mean or median baseline LDL-C level), despite stable statin therapy, of about 40 mg/dl to about 115 or about 40 to about 100 mg/dl.

In one embodiment, the subject or subject group being treated in accordance with methods of the invention is on concomitant statin therapy, for example atorvastatin, rosuvastatin or simvastatin therapy (with or without ezetimibe). In another embodiment, the subject is on concomitant stable statin therapy at time of initiation of ultra-pure EPA therapy.

In one embodiment, the subject or subject group being treated in accordance with methods of the invention has previously been treated with Lovaza® and has experienced an increase in, or no decrease in, LDL-C levels and/or non-HDL-C levels. In one such embodiment, Lovaza® therapy is discontinued and replaced by a method of the present invention

In another embodiment, the subject or subject group being treated in accordance with methods of the invention has a body mass index (BMI or mean BMI) of not more than about 45 kg/m².

In one embodiment, the invention provides a method of lowering triglycerides in a subject on stable statin therapy having baseline fasting triglycerides of about 200 mg/dl to about 500 mg/dl, the method comprising administering to the subject a pharmaceutical composition comprising about 1 g to about 4 g of EPA, about 2 g to about 4 g of EPA, or about 3.5 g to about 4 g of EPA, wherein upon administering the composition to the subject daily for a period of about 12 weeks the subject exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% lower fasting triglycerides than a control subject maintained on stable statin therapy (and optionally placebo matching the EPA) without concomitant EPA for a period of about 12 weeks, wherein the control subject also has baseline fasting triglycerides of about 200 mg/dl to about 500 mg/dl. The term “stable statin therapy” herein means that the subject, subject group, control subject or control subject group in question has been taking a stable daily dose of a statin (e.g. atorvastatin, rosuvastatin or simvastatin) for at least 4 weeks prior to the baseline fasting triglyceride measurement (the “qualifying period”). For example, a subject or control subject on stable statin therapy would receive a constant daily (i.e. the same dose each day) statin dose for at least 4 weeks immediately prior to baseline fasting triglyceride measurement. In one embodiment, the subject's and control subject's LDL-C is maintained between about 40 mg/dl and about 115 mg/dl or about 40 mg/dl to about 100 mg/dl during the qualifying period. The subject and control subject are then continued on their stable statin dose for the 12 week period post baseline.

In one embodiment, the statin is administered to the subject and the control subject in an amount of about 1 mg to about 500 mg, about 5 mg to about 200 mg, or about 10 mg to about 100 mg, for example about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg; about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, or about 500 mg. In another embodiment, the subject (and optionally the control subject) has a baseline LDL-C level, despite stable statin therapy, of about 40 mg/dl to about 115 mg/dl or about 40 mg/dl to about 100 mg/dl. In another embodiment, the subject and/or control subject has a body mass index (BMI; or mean BMI) of not more than about 45 kg/m².

In another embodiment, the invention provides a method of lowering triglycerides in a subject group on stable statin therapy having mean baseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl, the method comprising administering daily to members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA per day, wherein upon administering the composition to the members of the subject group daily for a period of about 12 weeks the subject group exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides than a control subject group maintained on stable statin therapy without concomitant EPA (optionally with matching placebo) for a period of about 12 weeks, wherein the control subject group also has mean baseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl. In a related embodiment, the stable statin therapy will be sufficient such that the subject group has a mean LDL-C level about at least about 40 mg/dl and not more than about 100 mg/dl or about 40 mg/dl to about 100 mg/dl for the 4 weeks immediately prior to the baseline fasting triglyceride measurement.

In another embodiment, the invention provides a method of lowering triglycerides in subject group on stable statin therapy and having a mean baseline fasting triglyceride level of about 200 mg/dl to about 499 mg/dl, the method comprising administering daily to members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to members of the subject group daily for a period of about 12 weeks the subject group exhibits: (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides by comparison with a control subject group maintained on stable statin therapy without concomitant ultra-pure EPA (optionally with matching placebo) for a period of about 12 weeks, and (b) no serum LDL-C increase, no statistically significant serum LDL-C increase, a serum LDL-C decrease, or the subject is statistically non-inferior to the control subjects (statin plus optional placebo) in regard to serum LDL-C elevation) no increase in mean serum LDL-C levels compared to baseline, wherein the control subject also has mean baseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl.

In another embodiment, the invention provides a method of lowering triglycerides in subject on stable statin therapy and having mean baseline fasting triglyceride level of about 200 mg/dl to about 499 mg/dl, the method comprising administering daily to the subject a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to the subject daily for a period of about 12 weeks the subject exhibits (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% lower fasting triglycerides by comparison with a control subject maintained on stable statin therapy without concomitant EPA for a period of about 12 weeks and (b) no increase in serum LDL-C levels compared to baseline, wherein the control subject also has baseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl.

In another embodiment, the invention provides a method of lowering triglycerides in subject group on stable statin therapy and having mean baseline fasting triglyceride level of about 200 mg/dl to about 499 mg/dl, the method comprising administering daily to members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to the members of the subject group daily for a period of about 12 weeks the subject group exhibits: (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides and (b) at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% lower mean serum LDL-C levels by comparison with a control subject group maintained on stable statin therapy without concomitant EPA (optionally with matching placebo) for a period of about 12 weeks, no serum LDL-C increase, no statistically significant serum LDL-C increase, no statistically significant serum LDL-C increase, a serum LDL-C decrease, or the subject group is statistically non-inferior to the control subject group (statin plus optional placebo) in regard to serum LDL-C elevation), wherein the control subject group also has mean baseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl.

In another embodiment, the invention provides a method of lowering triglycerides in subject group on stable statin therapy and having mean baseline fasting triglyceride level of about 200 mg/dl to about 499 mg/dl, the method comprising administering daily to members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to the members of the subject group daily for a period of about 12 weeks the subject group exhibits (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides and (b) at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% lower mean serum LDL-C levels by comparison with a control subject group maintained on stable statin therapy without concomitant EPA (optionally with matching placebo) for a period of about 12 weeks, no serum LDL-C increase, no statistically significant serum LDL-C increase, no statistically significant serum LDL-C increase, a serum LDL-C decrease, or the subject group is statistically non-inferior to the control subject group (statin plus optional placebo) in regard to serum LDL-C elevation), wherein the control subject group also has mean baseline fasting triglycerides of about 200 mg/dl to about 499 mg/dl.

In another embodiment, the subject or subject group being treated has a baseline triglyceride level (or median baseline triglyceride level in the case of a subject group), fed or fasting, of at least about 300 mg/dl, at least about 400 mg/dl, at least about 500 mg/dl, at least about 600 mg/dl, at least about 700 mg/dl, at least about 800 mg/dl, at least about 900 mg/dl, at least about 1000 mg/dl, at least about 1100 mg/dl, at least about 1200 mg/dl, at least about 1300 mg/dl, at least about 1400 mg/dl, or at least about 1500 mg/dl, for example about 400 mg/dl to about 2500 mg/dl, about 450 mg/dl to about 2000 mg/dl or about 500 mg/dl to about 1500 mg/dl.

In another embodiment, the invention provides a method of lowering triglycerides in a subject group having mean baseline fasting triglycerides of at least about 500 mg/dl, the method comprising administering daily to members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA per day, wherein upon administering the composition to the members of the subject group daily for a period of about 12 weeks the subject group exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides than a control subject group not receiving EPA (optionally with matching placebo) for a period of about 12 weeks, wherein the control subject group also has mean baseline fasting triglycerides of at least about 500 mg/dl. In a related embodiment, the members of the subject group and/or the control subject group also receive statin therapy, such as stable statin therapy.

In another embodiment, the invention provides a method of lowering triglycerides in a subject or subject group having mean baseline fasting triglycerides of at least about 500 mg/dl, the method comprising administering daily to the subject or the members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to members of the subject group daily for a period of about 12 weeks the subject group exhibits: (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides by comparison with a control subject group not receiving EPA (optionally with matching placebo) for a period of about 12 weeks, and (b) no serum LDL-C increase, no statistically significant serum LDL-C increase, a serum LDL-C decrease, or the subject is statistically non-inferior to the control subjects (statin plus optional placebo) in regard to serum LDL-C elevation) no increase in mean serum LDL-C levels compared to baseline, wherein the control subject also has mean baseline fasting triglycerides of at least about 500 mg/dl.

In another embodiment, the invention provides a method of lowering triglycerides in a subject or a subject group having mean baseline fasting triglycerides of at least about 500 mg/dl, the method comprising administering daily to the subject or subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to the subject daily for a period of about 12 weeks the subject exhibits (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% lower fasting triglycerides by comparison with a control subject not receiving EPA for a period of about 12 weeks and (b) no increase in serum LDL-C levels compared to baseline, wherein the control subject also has baseline fasting triglycerides of at least about 500 mg/dl.

In another embodiment, the invention provides a method of lowering triglycerides in a subject or subject group having mean baseline fasting triglycerides of at least about 500 mg/dl, the method comprising administering daily to the subject or members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to the members of the subject group daily for a period of about 12 weeks the subject group exhibits: (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides and (b) at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% lower mean serum LDL-C levels by comparison with a control subject group not receiving EPA (optionally with matching placebo) for a period of about 12 weeks, no serum LDL-C increase, no statistically significant serum LDL-C increase, no statistically significant serum LDL-C increase, a serum LDL-C decrease, or the subject group is statistically non-inferior to the control subject group (statin plus optional placebo) in regard to serum LDL-C elevation), wherein the control subject group also has mean baseline fasting triglycerides of at least about 500 mg/dl.

In another embodiment, the invention provides a method of lowering triglycerides in a subject or subject group having mean baseline fasting triglycerides of at least about 500 mg/dl, the method comprising administering daily to members of the subject group a pharmaceutical composition comprising about 1 g to about 4 g of EPA, wherein upon administering the composition to the members of the subject group daily for a period of about 12 weeks the subject group exhibits (a) at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% lower mean fasting triglycerides and (b) at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% lower mean serum LDL-C levels by comparison with a control subject group not receiving EPA (optionally with matching placebo) for a period of about 12 weeks, no serum LDL-C increase, no statistically significant serum LDL-C increase, no statistically significant serum LDL-C increase, a serum LDL-C decrease, or the subject group is statistically non-inferior to the control subject group (statin plus optional placebo) in regard to serum LDL-C elevation), wherein the control subject group also has mean baseline fasting triglycerides of at least about 500 mg/dl.

In another embodiment, the subject or subject group being treated in accordance with methods of the invention exhibits a fasting baseline absolute plasma level of free total fatty acid (or mean thereof) not greater than about 300 nmol/ml, not greater than about 250 nmol/ml, not greater than about 200 nmol/ml, not greater than about 150 nmol/ml, not greater than about 100 nmol/ml, or not greater than about 50 nmol/ml.

In another embodiment, the subject or subject group being treated in accordance with methods of the invention exhibits a fasting baseline absolute plasma level of free EPA (or mean thereof in the case of a subject group) not greater than about 0.70 nmol/ml, not greater than about 0.65 nmol/ml, not greater than about 0.60 nmol/ml, not greater than about 0.55 nmol/ml, not greater than about 0.50 nmol/ml, not greater than about 0.45 nmol/ml, or not greater than about 0.40 nmol/ml. In another embodiment, the subject or subject group being treated in accordance with methods of the invention exhibits a baseline fasting plasma level (or mean thereof) of free EPA, expressed as a percentage of total free fatty acid, of not more than about 3%, not more than about 2.5%, not more than about 2%, not more than about 1.5%, not more than about 1%, not more than about 0.75%, not more than about 0.5%, not more than about 0.25%, not more than about 0.2% or not more than about 0.15%. In one such embodiment, free plasma EPA and/or total fatty acid levels are determined prior to initiating therapy.

In another embodiment, the subject or subject group being treated in accordance with methods of the invention exhibits a fasting baseline absolute plasma level of free EPA (or mean thereof) not greater than about 1 nmol/ml, not greater than about 0.75 nmol/ml, not greater than about 0.50 nmol/ml, not greater than about 0.4 nmol/ml, not greater than about 0.35 nmol/ml, or not greater than about 0.30 nmol/ml.

In another embodiment, the subject or subject group being treated in accordance with methods of the invention exhibits a fasting baseline plasma, serum or red blood cell membrane EPA level not greater than about 150 μg/ml, not greater than about 125 μg/ml, not greater than about 100 μg/ml, not greater than about 95 μg/ml, not greater than about 75 μg/ml, not greater than about 60 μg/ml, not greater than about 50 μg/ml, not greater than about 40 μg/ml, not greater than about 30 μg/ml, or not greater than about 25 μg/ml.

In another embodiment, methods of the present invention comprise a step of measuring the subject's (or subject group's mean) baseline lipid profile prior to initiating therapy. In another embodiment, methods of the invention comprise the step of identifying a subject or subject group having one or more of the following: baseline non-HDL-C value (or mean) of about 200 mg/dl to about 400 mg/dl, for example at least about 210 mg/dl, at least about 220 mg/dl, at least about 230 mg/dl, at least about 240 mg/dl, at least about 250 mg/dl, at least about 260 mg/dl, at least about 270 mg/dl, at least about 280 mg/dl, at least about 290 mg/dl, or at least about 300 mg/dl; baseline total cholesterol value (or mean) of about 250 mg/dl to about 400 mg/dl, for example at least about 260 mg/dl, at least about 270 mg/dl, at least about 280 mg/dl or at least about 290 mg/dl; baseline VLDL-C value (or mean) of about 140 mg/dl to about 200 mg/dl, for example at least about 150 mg/dl, at least about 160 mg/dl, at least about 170 mg/dl, at least about 180 mg/dl or at least about 190 mg/dl; baseline HDL-C value (or mean) of about 10 to about 100 mg/dl, for example not more than about 90 mg/dl not, not more than about 80 mg/dl, not more than about 70 mg/dl, not more than about 60 mg/dl, not more than about 60 mg/dl, not more than about 50 mg/dl, not more than about 40 mg/dl, not more than about 35 mg/dl, not more than about 30 mg/dl, not more than about 25 mg/dl, not more than about 20 mg/dl, or not more than about 15 mg/dl; and/or baseline LDL-C value (or mean) of about 30 to about 300 mg/dl, for example not less than about 30 mg/dl, not less than about 40 mg/dl, not less than about 50 mg/dl, not less than about 60 mg/dl, not less than about 70 mg/dl, not less than about 90 mg/dl, not less than about 100 mg/dl, not less than about 110 mg/dl, not less than about 120 mg/dl, not less than about 130 mg/dl, not less than about 140 mg/dl, not less than about 150 mg/dl, not less than about 160 mg/dl, not less than about 170 mg/dl, not less than about 180 mg/dl, not less than about 190 mg/dl, not less than about 200 mg/dl, not less than about 210 mg/dl, not less than about 220 mg/dl, not less than about 230 mg/dl, not less than about 240 mg/dl, not less than about 250 mg/dl, not less than about 260 mg/dl, not less than about 270 mg/dl, not less than about 280 mg/dl, not less than about 290 mg/dl, or not less than about 300 mg/dl.

In a related embodiment, upon treatment in accordance with the present invention, for example over a period of about 1 to about 200 weeks, about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about 50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1 to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks, about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, the subject or subject group exhibits one or more of the following outcomes:

(a) reduced triglyceride levels compared to baseline or placebo control (e.g. a subject on stable statin plus placebo matching the EPA treatment group);

(b) reduced Apo B levels compared to baseline or placebo control;

(c) increased HDL-C levels compared to baseline or placebo control;

(d) no increase in LDL-C levels compared to baseline or placebo control;

(e) a reduction in LDL-C levels compared to baseline or placebo control;

(f) a reduction in non-HDL-C levels compared to baseline or placebo control;

(g) a reduction in VLDL levels compared to baseline or placebo control;

(h) an increase in apo A-I levels compared to baseline or placebo control;

(i) an increase in apo A-I/apo B ratio compared to baseline or placebo control;

(j) a reduction in lipoprotein A levels compared to baseline or placebo control;

(k) a reduction in LDL particle number compared to baseline or placebo control;

(l) an increase in LDL size compared to baseline or placebo control;

(m) a reduction in remnant-like particle cholesterol compared to baseline or placebo control;

(n) a reduction in oxidized LDL compared to baseline or placebo control;

(o) no change or a reduction in fasting plasma glucose (FPG) compared to baseline or placebo control;

(p) a reduction in hemoglobin A_(1c) (HbA_(1c)) compared to baseline or placebo control;

(q) a reduction in homeostasis model insulin resistance compared to baseline or placebo control;

(r) a reduction in lipoprotein associated phospholipase A2 compared to baseline or placebo control;

(s) a reduction in intracellular adhesion molecule-1 compared to baseline or placebo control;

(t) a reduction in interleukin-6 compared to baseline or placebo control;

(u) a reduction in plasminogen activator inhibitor-1 compared to baseline or placebo control;

(v) a reduction in high sensitivity C-reactive protein (hsCRP) compared to baseline or placebo control;

(w) an increase in serum or plasma EPA compared to baseline or placebo control;

(x) an increase in red blood cell membrane EPA compared to baseline or placebo control;

(y) a reduction or increase in one or more of serum and/or red blood cell content of docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), arachidonic acid (AA), palmitic acid (PA), stearidonic acid (SA) or oleic acid (OA) compared to baseline or placebo control;

(z) a reduction in the ratio of arachidonic acid to EPA (“AA/EPA”) in serum, plasma and/or RBCs compared to baseline or placebo control;

(aa) an increase in the ratio of omega-3 fatty acids to omega-6 fatty acids in serum, plasma, and/or RBCs compared to baseline or placebo control;

(bb) a decrease in the ratio of omega-6 fatty acids to total fatty acids in serum, plasma, and/or RBCs compared to baseline or placebo control; and/or

(cc) an increase in the ratio of omega-3 fatty acids to total fatty acids in serum, plasma, and/or RBCs compared to baseline or placebo control.

In one embodiment, methods of the present invention comprise measuring baseline levels of one or more markers set forth in (a)-(cc) above prior to dosing the subject or subject group. In another embodiment, the methods comprise administering a composition as disclosed herein to the subject after baseline levels of one or more markers set forth in (a)-(cc) are determined, and subsequently taking an additional measurement of said one or more markers.

In another embodiment, upon treatment with a composition of the present invention, for example over a period of about 1 to about 200 weeks, about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about 50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1 to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks, about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, the subject or subject group exhibits any 2 or more of, any 3 or more of, any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of, any 8 or more of, any 9 or more of, any 10 or more of, any 11 or more of, any 12 or more of, any 13 or more of, any 14 or more of, any 15 or more of, any 16 or more of, any 17 or more of, any 18 or more of, any 19 or more of, any 20 or more of, any 21 or more of, any 22 or more of, any 23 or more of, any 24 or more of, any 25 or more of, any 26 or more of, any 27 or more of, any 28 or more of, or all 29 of outcomes (a)-(cc) described immediately above.

In another embodiment, upon treatment with a composition of the present invention, the subject or subject group exhibits one or more of the following outcomes:

(a) a reduction in triglyceride level of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% or at least about 75% (actual % change or median % change) as compared to baseline or placebo control (e.g. a subject on statin and placebo matching the EPA treatment group);

(b) a less than 30% increase, less than 20% increase, less than 10% increase, less than 5% increase or no increase in non-HDL-C levels or a reduction in non-HDL-C levels of at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% or at least about 75% (actual % change or median % change) as compared to baseline or placebo control;

(c) substantially no change in HDL-C levels, no change in HDL-C levels, or an increase in HDL-C levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% or at least about 75% (actual % change or median % change) as compared to baseline or placebo control;

(d) a less than 60% increase, less than 50% increase, less than 40% increase, less than 30% increase, less than 20% increase, less than 10% increase, less than 5% increase or no increase in LDL-C levels, or a reduction in LDL-C levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 55% or at least about 75% (actual % change or median % change) as compared to baseline or placebo control;

(e) a decrease in Apo B levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% or at least about 75% (actual % change or median % change) as compared to baseline or placebo control;

(f) a reduction in VLDL levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(g) an increase in apo A-I levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(h) an increase in apo A-I/apo B ratio of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(i) a reduction in lipoprotein (a) levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(j) a reduction in mean LDL particle number of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(k) an increase in mean LDL particle size of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(l) a reduction in remnant-like particle cholesterol of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(m) a reduction in oxidized LDL of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(n) substantially no change, no statistically significant change, or a reduction in fasting plasma glucose (FPG) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(o) substantially no change, no statistically significant change, a reduction in hemoglobin A_(1c) (HbA_(1c)) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% (actual % change or median % change) compared to baseline or placebo control;

(p) a reduction in homeostasis model index insulin resistance of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(q) a reduction in lipoprotein associated phospholipase A2 of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(r) a reduction in intracellular adhesion molecule-1 of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(s) a reduction in interleukin-6 of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(t) a reduction in plasminogen activator inhibitor-1 of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(u) a reduction in high sensitivity C-reactive protein (hsCRP) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 100% (actual % change or median % change) compared to baseline or placebo control;

(v) an increase in serum, plasma and/or RBC EPA of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 200%, at least about 220%, at least about 223%, about 223.6%, 223.6%, at least about 230%, about 230.7%, 230.7%, at least about 250%, at least about 257%, about 257.3%, 257.3%, about 268%, 268.2% about 290%, 290.4%, 297.7%, about 298%, at least about 300%, at least about 345%, about 345.6%, 345.6%, at least about 400%, about 402%, 402.3%, at least about 450%, at least about 460%, at least about 462%, about 462.3%, 462.3%, at least about 480%, about 489.6%, 489.6%, about 490%, at least about 500%, at least about 600%, about 619%, 619.2%, at least about 645%, about 645.9%, about 646%, at least about 649%, about 649.7%, 649.7%, about 650%, at least about 698%, about 698.9%, about 699%, at least about 700%, about 790%, 792%, or at least about 800% (actual % change or median % change) compared to baseline or placebo control;

(w) an increase in serum phospholipid and/or red blood cell membrane EPA of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, r at least about 50%, at least about 100%, at least about 200%, or at least about 400% (actual % change or median % change) compared to baseline or placebo control;

(x) a reduction or increase in one or more of serum phospholipid and/or red blood cell DHA, DPA, AA, PA and/or OA of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% or at least about 75% (actual % change or median % change) compared to baseline or placebo control;

(y) a reduction in total cholesterol of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% or at least about 75% (actual % change or median % change) compared to baseline or placebo control;

(z) a reduction in the ratio of arachidonic acid to EPA (“AA/EPA”) in serum, plasma and/or RBCs of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, about 75.4%, at least about 80%, at least about 83%, about 83.7%, 83.7%, about 83.9%, 83.9%, about 84%, at least about 84%, about 84.2%, 84.2%, about 84.8%, 84.8%, about 85%, at least about 85%, at least about 86%, about 86.5%, about 87%, about 87.1%, about 87.8%, 87.8%, about 88%, 88.4%, at least about 90%, about 90.3%, at least about 95%, about 98.5%, about 91%, 91.1%, about 99%, 99.4%, at least about 100%, about 102%, 102%, at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 135%, at least about 140%, at least about 145%, or at least about 150% (actual % or median % change) compared to baseline or placebo control;

(aa) no significant change in the ratio of omega-3 fatty acids to omega-6 fatty acids in serum, plasma, and/or RBCs, or an increase in the ratio of omega-3 fatty acids to omega-6 fatty acids in serum, plasma, and/or RBCs of at least about 50%, at least about 54%, about 54.5%, 54.5%, about 55%, at least about 55%, at least about 60%, about 60.4%, about 64.3%, at least about 65%, about 65%, 65.5%, about 66%, at least about 70%, at least about 75%, 76.8%, about 77%, at least about 80%, at least about 85%, at least about 90%, about 90.9%, 90.9%, about 91%, at least about 95%, at least about 100%, at least about 105%, 106.9%, about 107%, at least about 110%, at least about 115%, at least about 120%, about 123.0%, at least about 125%, at least about 130%, at least about 135%, at least about 140%, at least about 145%, 145.8%, about 146%, at least about 150%, at least about 155%, at least about 160%, at least about 165%, about 165.9%, at least about 170%, at least about 175%, at least about 180%, at least about 185%, at least about 190%, at least about 195%, or at least about 200% (actual % or median % change) compared to baseline or placebo control;

(bb) a decrease in the ratio of omega-6 fatty acids to total fatty acids in serum, plasma, and/or RBCs of at least about 4%, about 4.6%, at least about 5%, at least about 6%, at least about 7%, about 7.4%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, about 11.3%, at least about 12%, at least about 13%, about 13%, 13.2%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, about 17%, 17.3%, at least about 18%, at least about 19%, about 19.7%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25% (actual % or median % change) compared to baseline or placebo control; and/or

(cc) an increase in the ratio of omega-3 fatty acids to total fatty acids in serum, plasma, and/or RBCs of at least about 40%, at least about 45%, at least about 50%, about 50.0%, about 53%, 53.4%, at least about 55%, at least about 60%, about 60.3%, at least about 65%, about 68%, 68.4%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, about 89.1%, at least about 90%, at least about 95%, at least about 100%, at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, 132.7%, about 133%, at least about 135%, at least about 140%, about 142.2%, at least about 145%, at least about 150%, at least about 155%, at least about 160%, at least about %, at least about 165%, at least about 170%, at least about 175%, at least about 180%, at least about 185%, at least about 190%, at least about 195%, at least about 200% (actual % or median % change) compared to baseline or placebo control.

In one embodiment, methods of the present invention comprise measuring baseline levels of one or more markers set forth in (a)-(cc) prior to dosing the subject or subject group. In another embodiment, the methods comprise administering a composition as disclosed herein to the subject after baseline levels of one or more markers set forth in (a)-(cc) are determined, and subsequently taking a second measurement of the one or more markers as measured at baseline for comparison thereto.

In another embodiment, upon treatment with a composition of the present invention, for example over a period of about 1 to about 200 weeks, about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about 50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1 to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks, about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, the subject or subject group exhibits any 2 or more of, any 3 or more of, any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of, any 8 or more of, any 9 or more of, any 10 or more of, any 11 or more of, any 12 or more of, any 13 or more of, any 14 or more of, any 15 or more of, any 16 or more of, any 17 or more of, any 18 or more of, any 19 or more of, any 20 or more of, any 21 or more of, any 22 or more of, any 23 or more of, any 24 or more of, any 25 or more of, any 26 or more of, any 27 or more of, any 28 or more of, or all 29 of outcomes (a)-(cc) described immediately above.

Parameters (a)-(cc) can be measured in accordance with any clinically acceptable methodology. For example, triglycerides, total cholesterol, HDL-C and fasting blood sugar can be sample from serum and analyzed using standard photometry techniques. VLDL-TG, LDL-C and VLDL-C can be calculated or determined using serum lipoprotein fractionation by preparative ultracentrifugation and subsequent quantitative analysis by refractometry or by analytic ultracentrifugal methodology. Apo A1, Apo B and hsCRP can be determined from serum using standard nephelometry techniques. Lipoprotein (a) can be determined from serum using standard turbidimetric immunoassay techniques. LDL particle number and particle size can be determined using nuclear magnetic resonance (NMR) spectrometry. Remnants lipoproteins and LDL-phospholipase A2 can be determined from EDTA plasma or serum and serum, respectively, using enzymatic immunoseparation techniques. Oxidized LDL, intercellular adhesion molecule-1 and interleukin-2 levels can be determined from serum using standard enzyme immunoassay techniques. These techniques are described in detail in standard textbooks, for example Tietz Fundamentals of Clinical Chemistry, 6^(th) Ed. (Burtis, Ashwood and Borter Eds.), WB Saunders Company.

In one embodiment, subjects fast for up to 12 hours prior to blood sample collection, for example about 10 hours.

In another embodiment, the subject being treated is in the highest risk category of Adult Treatment Panel (ATP) III Classification of LDL, Total, and HDL Cholesterol (mg/dL) (e.g. CHD or CHD Risk Equivalents (10-year risk >20%)). In another embodiment, the subject is in the ATP III Multiple (2+) risk factor category.

In one embodiment, the invention provides a method of lowering triglycerides in a subject in the highest risk category of Adult Treatment Panel (ATP) III Classification of LDL, Total, and HDL Cholesterol (mg/dL) (e.g. CHD or CHD Risk Equivalents (10-year risk >20%)). In another embodiment, the subject is in the ATP III Multiple (2+) risk factor category. In another embodiment, the method includes a step of identifying a subject in the ATP III Multiple (2+) risk factor category prior to administering ultra-pure E-EPA to the subject.

In another embodiment, the present invention provides a method of treating or preventing primary hypercholesterolemia and/or mixed dyslipidemia (Fredrickson Types IIa and III)) in a patient in need thereof, comprising administering to the patient one or more compositions as disclosed herein. In a related embodiment, the present invention provides a method of reducing triglyceride levels in a subject or subjects when treatment with a statin or niacin extended-release monotherapy is considered inadequate (Frederickson type IV hyperlipidemia).

In another embodiment, the present invention provides a method of treating or preventing risk of recurrent nonfatal myocardial infarction in a patient with a history of myocardial infarction, comprising administering to the patient one or more compositions as disclosed herein.

In another embodiment, the present invention provides a method of slowing progression of or promoting regression of atherosclerotic disease in a patient in need thereof, comprising administering to a subject in need thereof one or more compositions as disclosed herein.

In another embodiment, the present invention provides a method of treating or preventing very high serum triglyceride levels (e.g. Types IV and V hyperlipidemia) in a patient in need thereof, comprising administering to the patient one or more compositions as disclosed herein.

In another embodiment, the present invention provides a method of treating subjects having very high serum triglyceride levels (e.g. greater than 1000 mg/dl or greater than 2000 mg/dl) and that are at risk of developing pancreatitis, comprising administering to the patient one or more compositions as disclosed herein.

In one embodiment, a composition of the invention is administered to a subject in an amount sufficient to provide a daily dose of EPA of about 1 mg to about 10,000 mg, 25 about 5000 mg, about 50 to about 3000 mg, about 75 mg to about 2500 mg, or about 100 mg to about 1000 mg, for example about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about 1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg, about 2050 mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150 mg, about 2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about 2275 mg, about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg, about 2400 mg, about 2425 mg, about 2450 mg, about 2475 mg, about 2500 mg, about 2525 mg, about 2550 mg, about 2575 mg, about 2600 mg, about 2625 mg, about 2650 mg, about 2675 mg, about 2700 mg, about 2725 mg, about 2750 mg, about 2775 mg, about 2800 mg, about 2825 mg, about 2850 mg, about 2875 mg, about 2900 mg, about 2925 mg, about 2950 mg, about 2975 mg, about 3000 mg, about 3025 mg, about 3050 mg, about 3075 mg, about 3100 mg, about 3125 mg, about 3150 mg, about 3175 mg, about 3200 mg, about 3225 mg, about 3250 mg, about 3275 mg, about 3300 mg, about 3325 mg, about 3350 mg, about 3375 mg, about 3400 mg, about 3425 mg, about 3450 mg, about 3475 mg, about 3500 mg, about 3525 mg, about 3550 mg, about 3575 mg, about 3600 mg, about 3625 mg, about 3650 mg, about 3675 mg, about 3700 mg, about 3725 mg, about 3750 mg, about 3775 mg, about 3800 mg, about 3825 mg, about 3850 mg, about 3875 mg, about 3900 mg, about 3925 mg, about 3950 mg, about 3975 mg, about 4000 mg, about 4025 mg, about 4050 mg, about 4075 mg, about 4100 mg, about 4125 mg, about 4150 mg, about 4175 mg, about 4200 mg, about 4225 mg, about 4250 mg, about 4275 mg, about 4300 mg, about 4325 mg, about 4350 mg, about 4375 mg, about 4400 mg, about 4425 mg, about 4450 mg, about 4475 mg, about 4500 mg, about 4525 mg, about 4550 mg, about 4575 mg, about 4600 mg, about 4625 mg, about 4650 mg, about 4675 mg, about 4700 mg, about 4725 mg, about 4750 mg, about 4775 mg, about 4800 mg, about 4825 mg, about 4850 mg, about 4875 mg, about 4900 mg, about 4925 mg, about 4950 mg, about 4975 mg, about 5000 mg, about 5050 mg, about 5100 mg, about 5150 mg, about 5200 mg, about 5250 mg, about 5300 mg, about 5350 mg, about 5400 mg, about 5450 mg, about 5500 mg, about 5550 mg, about 5600 mg, about 5650 mg, about 5700 mg, about 5750 mg, about 5800 mg, about 5850 mg, about 5900 mg, about 5950 mg, about 6000 mg, about 6050 mg, about 6100 mg, about 6150 mg, about 6200 mg, about 6250 mg, about 6300 mg, about 6350 mg, about 6400 mg, about 6450 mg, about 6500 mg, about 6550 mg, about 6600 mg, about 6650 mg, about 6700 mg, about 6750 mg, about 6800 mg, about 6850 mg, about 6900 mg, about 6950 mg, about 7000 mg, about 7050 mg, about 7100 mg, about 7150 mg, about 7200 mg, about 7250 mg, about 7300 mg, about 7350 mg, about 7400 mg, about 7450 mg, about 7500 mg, about 7550 mg, about 7600 mg, about 7650 mg, about 7700 mg, about 7750 mg, about 7800 mg, about 7850 mg, about 7900 mg, about 7950 mg, about 8000 mg, about 8050 mg, about 8100 mg, about 8150 mg, about 8200 mg, about 8250 mg, about 8300 mg, about 8350 mg, about 8400 mg, about 8450 mg, about 8500 mg, about 8550 mg, about 8600 mg, about 8650 mg, about 8700 mg, about 8750 mg, about 8800 mg, about 8850 mg, about 8900 mg, about 8950 mg, about 9000 mg, about 9050 mg, about 9100 mg, about 9150 mg, about 9200 mg, about 9250 mg, about 9300 mg, about 9350 mg, about 9400 mg, about 9450 mg, about 9500 mg, about 9550 mg, about 9600 mg, about 9650 mg, about 9700 mg, about 9750 mg, about 9800 mg, about 9850 mg, about 9900 mg, about 9950 mg, or about 10,000 mg.

In another embodiment, any of the methods disclosed herein are used in treatment of a subject or subjects that consume a traditional Western diet. In one embodiment, the methods of the invention include a step of identifying a subject as a Western diet consumer or prudent diet consumer and then treating the subject if the subject is deemed a Western diet consumer. The term “Western diet” herein refers generally to a typical diet consisting of, by percentage of total calories, about 45% to about 50% carbohydrate, about 35% to about 40% fat, and about 10% to about 15% protein. A Western diet may alternately or additionally be characterized by relatively high intakes of red and processed meats, sweets, refined grains, and desserts, for example more than 50%, more than 60% or more or 70% of total calories come from these sources.

In another embodiment, any of the methods disclosed herein are used in treatment of a subject or subjects that consume less than (actual or average) about 150 g, less than about 125 g, less than about 100 g, less than about 75 g, less than about 50 g, less than about 45 g, less than about 40 g, less than about 35 g, less than about 30 g, less than about 25 g, less than about 20 g or less than about 15 g of fish per day.

In another embodiment, any of the methods disclosed herein are used in treatment of a subject or subjects that consume less than (actual or average) about 10 g, less than about 9 g, less than about 8 g, less than about 7 g, less than about 6 g, less than about 5 g, less than about 4 g, less than about 3 g, less than about 2 g per day of omega-3 fatty acids from dietary sources.

In another embodiment, any of the methods disclosed herein are used in treatment of a subject or subjects that consume less than (actual or average) about 2.5 g, less than about 2 g, less than about 1.5 g, less than about 1 g, less than about 0.5 g, less than about 0.25 g, or less than about 0.2 g per day of EPA and DHA (combined) from dietary sources.

In one embodiment, compositions useful in various embodiments of the invention comprise a polyunsaturated fatty acid as an active ingredient. In another embodiment, such compositions comprise EPA as an active ingredient. The term “EPA” as used herein refers to eicosapentaenoic acid (e.g. eicosa-5,8,11,14,17-pentaenoic acid) and/or a pharmaceutically acceptable ester, derivative, conjugate or salt thereof, or mixtures of any of the foregoing.

In one embodiment, the EPA comprises all-cis eicosa-5,8,11,14,17-pentaenoic acid. In another embodiment, the EPA is in the form of an eicosapentaenoic acid ester. In another embodiment, the EPA comprises a C₁-C₅ alkyl ester of EPA. In another embodiment, the EPA comprises eicosapentaenoic acid ethyl ester, eicosapentaenoic acid methyl ester, eicosapentaenoic acid propyl ester, or eicosapentaenoic acid butyl ester. In still another embodiment, the EPA comprises all-cis eicosa-5,8,11,14,17-pentaenoic acid ethyl ester.

In still other embodiments, the EPA comprises ethyl-EPA, lithium EPA, mono, di- or triglyceride EPA or any other ester or salt of EPA, or the free acid form of EPA. The EPA may also be in the form of a 2-substituted derivative or other derivative which slows down its rate of oxidation but does not otherwise change its biological action to any substantial degree.

The term “pharmaceutically acceptable” in the present context means that the substance in question does not produce unacceptable toxicity to the subject or interaction with other components of the composition.

In one embodiment, EPA present in a composition suitable for use according to the invention comprises ultra-pure EPA. The term “ultra-pure” as used herein with respect to EPA refers to a composition comprising at least 96% by weight EPA (as the term “EPA” is defined and exemplified herein). Ultra-pure EPA can comprise even higher purity EPA, for example at least 97% by weight EPA, at least 98% by weight EPA or at least 99% by weight EPA, wherein the EPA is any form of EPA as set forth herein. Ultra-pure EPA can further be defined (e.g. impurity profile) by any of the description of EPA provided herein.

In some embodiments, EPA is present in a composition in an amount of about 50 mg to about 5000 mg, about 75 mg to about 2500 mg, or about 100 mg to about 1000 mg, for example about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about 1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg, about 2050 mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150 mg, about 2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about 2275 mg, about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg, about 2400 mg, about 2425 mg, about 2450 mg, about 2475 mg, or about 2500 mg, about 2525 mg, about 2550 mg, about 2575 mg, about 2600 mg, about 2625 mg, about 2650 mg, about 2675 mg, about 2700 mg, about 2725 mg, about 2750 mg, about 2775 mg, about 2800 mg, about 2825 mg, about 2850 mg, about 2875 mg, about 2900 mg, about 2925 mg, about 2950 mg, about 2975 mg, about 3000 mg, about 3025 mg, about 3050 mg, about 3075 mg, about 3100 mg, about 3125 mg, about 3150 mg, about 3175 mg, about 3200 mg, about 3225 mg, about 3250 mg, about 3275 mg, about 3300 mg, about 3325 mg, about 3350 mg, about 3375 mg, about 3400 mg, about 3425 mg, about 3450 mg, about 3475 mg, about 3500 mg, about 3525 mg, about 3550 mg, about 3575 mg, about 3600 mg, about 3625 mg, about 3650 mg, about 3675 mg, about 3700 mg, about 3725 mg, about 3750 mg, about 3775 mg, about 3800 mg, about 3825 mg, about 3850 mg, about 3875 mg, about 3900 mg, about 3925 mg, about 3950 mg, about 3975 mg, about 4000 mg, about 4025 mg, about 4050 mg, about 4075 mg, about 4100 mg, about 4125 mg, about 4150 mg, about 4175 mg, about 4200 mg, about 4225 mg, about 4250 mg, about 4275 mg, about 4300 mg, about 4325 mg, about 4350 mg, about 4375 mg, about 4400 mg, about 4425 mg, about 4450 mg, about 4475 mg, about 4500 mg, about 4525 mg, about 4550 mg, about 4575 mg, about 4600 mg, about 4625 mg, about 4650 mg, about 4675 mg, about 4700 mg, about 4725 mg, about 4750 mg, about 4775 mg, about 4800 mg, about 4825 mg, about 4850 mg, about 4875 mg, about 4900 mg, about 4925 mg, about 4950 mg, about 4975 mg, or about 5000 mg.

In various embodiments, one or more antioxidants can be present in the EPA (e.g. E-EPA or ultra pure E-EPA). Non-limiting examples of suitable antioxidants include tocopherol, lecithin, citric acid and/or ascorbic acid. One or more antioxidants, if desired, are typically present in the EPA in an amount of about 0.01% to about 0.1%, by weight, or about 0.025% to about 0.05%, by weight.

In one embodiment, a composition of the invention contains not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, or not more than about 0.5%, by weight of total fatty acids, docosahexaenoic acid (“DHA”) or derivative thereof such as E-DHA, if any. In another embodiment, a composition of the invention contains substantially no docosahexaenoic acid or derivative thereof such as E-DHA. In still another embodiment, a composition of the invention contains no docosahexaenoic acid or E-DHA.

In another embodiment, EPA represents at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%, by weight, of all fatty acids present in a composition useful in accordance with the invention.

In another embodiment, a composition of the invention contains less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.25%, by weight of the total composition or by weight of the total fatty acid content, of any fatty acid other than EPA, or derivative thereof. Illustrative examples of a “fatty acid other than EPA” include linolenic acid (LA) or derivative thereof such as ethyl-linolenic acid, arachidonic acid (AA) or derivative thereof such as ethyl-AA, docosahexaenoic acid (DHA) or derivative thereof such as ethyl-DHA, alpha-linolenic acid (ALA) or derivative thereof such as ethyl-ALA, stearadonic acid (STA) or derivative thereof such as ethyl-SA, eicosatrienoic acid (ETA) or derivative thereof such as ethyl-ETA and/or docosapentaenoic acid (DPA) or derivative thereof such as ethyl-DPA.

In another embodiment, a composition of the invention has one or more of the following features: (a) eicosapentaenoic acid ethyl ester represents at least 96%, at least 97%, or at least 98%, by weight, of all fatty acids present in the composition; (b) the composition contains not more than 4%, not more than 3%, or not more than 2%, by weight, of total fatty acids other than eicosapentaenoic acid ethyl ester; (c) the composition contains not more than 0.6%, 0.5%, 0.4% or 0.3% of any individual fatty acid other than eicosapentaenoic acid ethyl ester; (d) the composition has a refractive index (20° C.) of about 1 to about 2, about 1.2 to about 1.8 or about 1.4 to about 1.5; (e) the composition has a specific gravity (20° C.) of about 0.8 to about 1.0, about 0.85 to about 0.95 or about 0.9 to about 0.92; (f) the composition contains not more than 20 ppm, 15 ppm or 10 ppm heavy metals, (g) the composition contains not more than 5 ppm, 4 ppm, 3 ppm, or 2 ppm arsenic, and/or (h) the composition has a peroxide value not more than 5, 4, 3, or 2 meq/kg.

In another embodiment, a composition useful in accordance with the invention comprises, consists essentially of or consists of at least 95% by weight ethyl eicosapentaenoate (EPA-E), about 0.2% to about 0.5% by weight ethyl octadecatetraenoate (ODTA-E), about 0.05% to about 0.25% by weight ethyl nonaecapentaenoate (NDPA-E), about 0.2% to about 0.45% by weight ethyl arachidonate (AA-E), about 0.3% to about 0.5% by weight ethyl eicosatetraenoate (ETA-E), and about 0.05% to about 0.32% ethyl heneicosapentaenoate (HPA-E). In another embodiment, the composition is present in a capsule shell. In still another embodiment, the capsule shell contains no chemically modified gelatin.

In another embodiment, compositions useful in accordance with the invention comprise, consist essentially of, or consist of at least 95%, 96% or 97%, by weight, ethyl eicosapentaenoate, about 0.2% to about 0.5% by weight ethyl octadecatetraenoate, about 0.05% to about 0.25% by weight ethyl nonaecapentaenoate, about 0.2% to about 0.45% by weight ethyl arachidonate, about 0.3% to about 0.5% by weight ethyl eicosatetraenoate, and about 0.05% to about 0.32% by weight ethyl heneicosapentaenoate. Optionally, the composition contains not more than about 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivative thereof such as ethyl-DHA. In one embodiment the composition contains substantially no or no amount of DHA or derivative thereof such as ethyl-DHA. The composition further optionally comprises one or more antioxidants (e.g. tocopherol) in an amount of not more than about 0.5% or not more than 0.05%. In another embodiment, the composition comprises about 0.05% to about 0.4%, for example about 0.2% by weight tocopherol. In another embodiment, about 500 mg to about 1 g of the composition is provided in a capsule shell. In another embodiment, the capsule shell contains no chemically modified gelatin.

In another embodiment, compositions useful in accordance with the invention comprise, consist essentially of, or consist of at least 96% by weight ethyl eicosapentaenoate, about 0.22% to about 0.4% by weight ethyl octadecatetraenoate, about 0.075% to about 0.20% by weight ethyl nonaecapentaenoate, about 0.25% to about 0.40% by weight ethyl arachidonate, about 0.3% to about 0.4% by weight ethyl eicosatetraenoate and about 0.075% to about 0.25% by weight ethyl heneicosapentaenoate. Optionally, the composition contains not more than about 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivative thereof such as ethyl-DHA. In one embodiment the composition contains substantially no or no amount of DHA or derivative thereof such as ethyl-DHA. The composition further optionally comprises one or more antioxidants (e.g. tocopherol) in an amount of not more than about 0.5% or not more than 0.05%. In another embodiment, the composition comprises about 0.05% to about 0.4%, for example about 0.2% by weight tocopherol. In another embodiment, the invention provides a dosage form comprising about 500 mg to about 1 g of the foregoing composition in a capsule shell. In one embodiment, the dosage form is a gel- or liquid-containing capsule and is packaged in blister packages of about 1 to about 20 capsules per sheet.

In another embodiment, compositions useful in accordance with the invention comprise, consist essentially of or consist of at least 96%, 97% or 98%, by weight, ethyl eicosapentaenoate, about 0.25% to about 0.38% by weight ethyl octadecatetraenoate, about 0.10% to about 0.15% by weight ethyl nonaecapentaenoate, about 0.25% to about 0.35% by weight ethyl arachidonate, about 0.31% to about 0.38% by weight ethyl eicosatetraenoate, and about 0.08% to about 0.20% by weight ethyl heneicosapentaenoate. Optionally, the composition contains not more than about 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivative thereof such as ethyl-DHA. In one embodiment the composition contains substantially no or no amount of DHA or derivative thereof such as ethyl-DHA. The composition further optionally comprises one or more antioxidants (e.g. tocopherol) in an amount of not more than about 0.5% or not more than 0.05%. In another embodiment, the composition comprises about 0.05% to about 0.4%, for example about 0.2% by weight tocopherol. In another embodiment, the invention provides a dosage form comprising about 500 mg to about 1 g of the foregoing composition in a capsule shell. In another embodiment, the capsule shell contains no chemically modified gelatin.

In another embodiment, the invention provides a method of increasing serum, plasma and/or red blood cell (RBC) EPA levels comprising administering a composition as described herein to a subject in need of such treatment. In one embodiment, upon orally administering a composition as set forth herein to a subject for a period of at least about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 42, about 45 or about 50 days, the subject exhibits at least about a 2-fold, at least about a 3-fold, at least about a 3.5-fold, at least about a 3.75-fold or at least about a 4-fold increase or change (final absolute EPA level divided by baseline EPA level) in serum, plasma and/or RBC EPA. In one embodiment, the method comprises a step of identifying a patient in need of an increase in serum, plasma and/or red blood cell (RBC) EPA prior to said administration step. In a related embodiment, the subject has a baseline EPA plasma, serum and/or RBC level not greater than about 50 μg/g. In another embodiment, the subject is provided with about 2 g to about 4 g per day of a composition as described herein. In another embodiment, upon administering the composition to the subject as per above, the subject exhibits a decrease in DHA, AA and/or DGLA plasma, serum and/or RBC levels. In another embodiment, upon administering the composition to the subject as per above, the subject exhibits an increase in DPA plasma, serum and/or RBC levels. In still another embodiment, upon administering the composition to the subject as per above, DHA plasma, serum and/or RBC levels decrease by at least 15%, DGLA plasma, serum and/or RBC levels decrease by at least 30%, AA plasma, serum and/or RBC levels decrease by at least 20%, and/or DPA plasma, serum and/or RBC levels increase by greater than 130%.

In another embodiment, the invention provides a method of increasing serum, plasma and/or red blood cell (RBC) EPA levels comprising administering a composition as described herein to a subject in need of increased serum, plasma and/or RBC EPA levels. In a related embodiment, upon administering the composition to the subject for a period of at least about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 42, about 45, or about 50 days, the subject exhibits at least about a 100%, at least about a 150%, at least about a 200%, at least about a 250%, at least about a 300%, at least about a 350% or at least about a 400% increase (change in EPA level from baseline divided by baseline EPA level) in plasma, serum and/or RBC EPA levels compared to baseline. In a related embodiment, the subject has a baseline EPA plasma, serum and/or RBC level not greater than about 50 μg/g. In another embodiment, the subject is provided with about 2 g to about 4 g per day of a composition as described herein. In another embodiment, upon administering the composition to the subject as per above, the subject exhibits a decrease in DHA, AA and/or DGLA plasma, serum and/or RBC levels. In another embodiment, upon administering the composition to the subject as per above, the subject exhibits an increase in DPA plasma, serum and/or RBC levels. In still another embodiment, upon administering the composition to the subject as per above, DHA plasma, serum and/or RBC levels decrease by at least 15%, DGLA plasma, serum and/or RBC levels decrease by at least 30%, AA plasma, serum and/or RBC levels decrease by at least 20%, and/or DPA plasma, serum and/or RBC levels increase by greater than 130%.

In a related embodiment, upon orally administering about 2 to about 4 g per day of a composition as described herein to a subject for a period of at least about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 days, the subject exhibits at least about a 10 μg/g increase, at least about a 15 μg/g increase, at least about a 20 μg/g increase, at least about a 25 μg/g increase, at least about a 30 μg/g increase, at least about a 35 μg/g increase, at least about a 40 μg/g increase, at least about a 45 μg/g increase, at least about a 50 μg/g increase, at least about a 75 μg/g increase, at least about a 100 μg/g increase, or at least about a 150 μg/g increase in serum, plasma and/or RBC EPA compared to baseline. In another embodiment, upon administering the composition to the subject as per above, the subject exhibits a decrease in DHA, AA and/or DGLA plasma, serum and/or RBC levels. In another embodiment, upon administering the composition to the subject as per above, the subject exhibits an increase in DPA plasma, serum and/or RBC levels. In still another embodiment, upon administering the composition to the subject as per above, DHA plasma, serum and/or RBC levels decrease by at least 15%, DGLA plasma, serum and/or RBC levels decrease by at least 30%, AA plasma, serum and/or RBC levels decrease by at least 20%, and/or DPA plasma, serum and/or RBC levels increase by greater than 130%.

In another embodiment, the subject has not been on an omega-3 fatty acid therapy or supplement for at least 2 weeks, 3 weeks, 4 weeks, 6 weeks or 12 weeks prior to initiating therapy as described herein.

In another embodiment, a composition as described herein is administered to a subject once or twice per day. In another embodiment, 1, 2, 3 or 4 capsules, each containing about 1 g (e.g., about 900 mg to about 1.1 g) of a composition as described herein are administered to a subject daily. In another embodiment, 1 or 2 capsules, each containing about 1 g of a composition as described herein, are administered to the subject in the morning, for example between about 5 am and about 11 am, and 1 or 2 capsules, each containing about 1 g of a composition as described herein, are administered to the subject in the evening, for example between about 5 pm and about 11 pm.

In one embodiment, a subject being treated in accordance with methods of the invention is not on fibrate or nitrate therapy.

In one embodiment, a subject being treated in accordance with methods of the invention is not otherwise on lipid-altering therapy, for example statin, fibrate, niacin and/or ezetimibe therapy.

In another embodiment, compositions useful in accordance with methods of the invention are orally deliverable. The terms “orally deliverable” or “oral administration” herein include any form of delivery of a therapeutic agent or a composition thereof to a subject wherein the agent or composition is placed in the mouth of the subject, whether or not the agent or composition is swallowed. Thus “oral administration” includes buccal and sublingual as well as esophageal administration. In one embodiment, the composition is present in a capsule, for example a soft gelatin capsule.

A composition for use in accordance with the invention can be formulated as one or more dosage units. The terms “dose unit” and “dosage unit” herein refer to a portion of a pharmaceutical composition that contains an amount of a therapeutic agent suitable for a single administration to provide a therapeutic effect. Such dosage units may be administered one to a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.

In another embodiment, the invention provides use of any composition described herein for treating moderate to severe hypertriglyceridemia in a subject in need thereof, comprising: providing a subject having a fasting baseline triglyceride level of about 500 mg/dl to about 1500 mg/dl and administering to the subject a pharmaceutical composition as described herein. In one embodiment, the composition comprises about 1 g to about 4 g of eicosapentaenoic acid ethyl ester, wherein the composition contains substantially no docosahexaenoic acid.

In one embodiment, compositions of the invention, upon storage in a closed container maintained at room temperature, refrigerated (e.g. about 5 to about 5-10° C.) temperature, or frozen for a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, exhibit at least about 90%, at least about 95%, at least about 97.5%, or at least about 99% of the active ingredient(s) originally present therein.

In one embodiment, the invention provides use of a composition as described herein in manufacture of a medicament for treatment of any of a cardiovascular-related disease. In another embodiment, the subject is diabetic.

In one embodiment, a composition as set forth herein is packaged together with instructions for using the composition to treat a cardiovascular disorder.

It will be appreciated that the pharmacological activity of the compositions of the invention can be demonstrated using standard pharmacological models that are known in the art. Furthermore, it will be appreciated that the disclosed compositions can be incorporated or encapsulated in a suitable polymer matrix or membrane for site-specific delivery, or can be functionalized with specific targeting agents capable of effecting site specific delivery. These techniques, as well as other drug delivery techniques, are well known in the art.

In one embodiment, a pharmaceutical composition of the present disclosure provides a mean total eicosapentaenoic acid, unesterified plasma eicosapentaenoic acid, or total eicosapentaenoic acid red blood cell C_(max) of about 0.5 μg/mL to about 500 μg/mL, for example about 105 μg/mL to about 204 μg/mL, about 235 μg/mL to about 460 μg/mL, about 0.5 μg/mL, about 0.6 μg/mL, about 0.7 μg/mL (e.g, about 0.66 μg/mL), about 1 μg/mL, about 1.4 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 31 μg/mL, about 35 μg/mL, about 38 μg/mL (e.g, about 37.6 μg/mL), about 40 μg/mL, about 42 μg/mL (e.g, about 42.3 μg/mL), about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 77 μg/mL (e.g, about 76.7 μg/mL), about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, about 100 μg/mL, about 105 μg/mL, about 110 μg/mL, about 115 μg/mL, about 120 μg/mL, about 125 μg/mL, about 130 μg/mL, about 135 μg/mL, about 140 μg/mL, about 145 μg/mL, about 150 μg/mL, about 155 μg/mL (e.g, about 154.9 μg/mL), about 160 μg/mL, about 165 μg/mL, about 170 μg/mL, about 175 μg/mL, about 180 μg/mL, about 185 μg/mL, about 190 μg/mL, about 195 μg/mL, about 200 μg/mL, about 205 μg/mL, about 210 μg/mL (e.g, about 210.5 μg/mL), about 215 μg/mL, about 220 μg/mL, about 225 μg/mL, about 230 μg/mL, about 233 μg/mL (e.g, about 232.8 μg/mL), about 235 μg/mL, about 240 μg/mL, about 245 μg/mL, about 250 μg/mL, about 255 μg/mL, about 260 μg/mL, about 265 μg/mL, about 270 μg/mL, about 275 μg/mL, about 280 μg/mL, about 285 μg/mL, about 290 μg/mL, about 295 μg/mL, about 300 μg/mL, about 305 μg/mL, about 310 μg/mL, about 315 μg/mL, about 320 μg/mL, about 325 μg/mL, about 330 μg/mL, about 335 μg/mL, about 340 μg/mL, about 345 μg/mL, about about 347 μg/mL (e.g., about 347.2 μg/mL), about 350 μg/mL, about 355 μg/mL, about 360 μg/mL, about 365 μg/mL, about 370 μg/mL, about 375 μg/mL, about 380 μg/mL, about 385 μg/mL, about 390 μg/mL, about 395 μg/mL, about 400 μg/mL, about 405 μg/mL, about 410 μg/mL, about 415 μg/mL, about 420 μg/mL, about 425 μg/mL, about 430 μg/mL, about 435 μg/mL, about 440 μg/mL, about 445 μg/mL, about 450 μg/mL, about 455 μg/mL, about 460 μg/mL, about 465 μg/mL, about 470 μg/mL, about 475 μg/mL, about 480 μg/mL, about 485 μg/mL, about 490 μg/mL, about 495 μg/mL, or about 500 μg/mL.

In one embodiment, a pharmaceutical composition of the present disclosure provides a mean total eicosapentaenoic acid, unesterified plasma eicosapentaenoic acid, or total eicosapentaenoic acid red blood cell AUC_(0-24h) of about 1 μg·h/mL to about 7000 μg·h/mL, for example about 1 μg·h/mL to about 20 μg·h/mL, about 500 μg·h/mL to about 1500 μg·h/mL, about 2500 μg·h/mL to about 7000 μg·h/mL, about 1 μg·h/mL, about 2 μg·h/mL, about 3 μg·h/mL, about 4 μg·h/mL, about 5 μg·h/mL, about 6 μg·h/mL, about 7 μg·h/mL, about 8 μg·h/mL, about 9 μg·h/mL, about 10 μg·h/mL, about 11 μg·h/mL, about 12 μg·h/mL, about 13 μg·h/mL, about 14 μg·h/mL, about 15 μg·h/mL, about 16 μg·h/mL, about 17 μg·h/mL, about 18 μg·h/mL, about 19 μg·h/mL, about 20 μg·h/mL, about 500 μg·h/mL, about 550 μg·h/mL, about 600 μg·h/mL, about 650 μg·h/mL, about 700 μg·h/mL, about 750 μg·h/mL, about 800 μg·h/mL, about 850 μg·h/mL, about 900 μg·h/mL, about 950 μg·h/mL about 1000 μg·h/mL, about 1050 μg·h/mL about 1100 μg·h/mL, about 1150 μg·h/mL about 1200 μg·h/mL about 1250 μg·h/mL, about 1300 μg·h/mL, about 1350 μg·h/mL about 1400 μg·h/mL about 1450 μg·h/mL about 1500 μg·h/mL, about 2500 μg·h/mL about 2550 μg·h/mL about 2600 μg·h/mL about 2650 μg·h/mL, about 2700 μg·h/mL about 2750 μg·h/mL about 2800 μg·h/mL about 2850 μg·h/mL, about 2900 μg·h/mL about 2950 μg·h/mL about 3000 μg·h/mL about 3050 μg·h/mL, about 3100 μg·h/mL about 3150 μg·h/mL about 3200 μg·h/mL, about 3250 μg·h/mL, about 3300 μg·h/mL about 3350 μg·h/mL about 3400 μg·h/mL about 3450 μg·h/mL, about 3500 μg·h/mL about 3550 μg·h/mL about 3600 μg·h/mL about 3650 μg·h/mL, about 3700 μg·h/mL about 3750 μg·h/mL about 3800 μg·h/mL about 3850 μg·h/mL, about 3900 μg·h/mL about 3950 μg·h/mL about 4000 μg·h/mL about 4050 μg·h/mL, about 4100 μg·h/mL about 4150 μg·h/mL about 4200 μg·h/mL, about 4250 μg·h/mL/mL, about 4300 μg·h/mL, about 4350 μg·h/mL, about 4400 μg·h/mL, about 4450 μg·h/mL, about 4500 μg·h/mL about 4550 μg·h/mL about 4600 μg·h/mL about 4650 μg·h/mL, about 4700 μg·h/mL about 4750 μg·h/mL about 4800 μg·h/mL about 4850 μg·h/mL, about 4900 μg·h/mL about 4950 μg·h/mL about 5000 μg·h/mL about 5050 μg·h/mL, about 5100 μg·h/mL about 5150 μg·h/mL about 5200 μg·h/mL, about 5250 μg·h/mL, about 5300 μg·h/mL about 5350 μg·h/mL about 5400 μg·h/mL about 5450 μg·h/mL, about 5500 μg·h/mL about 5550 μg·h/mL about 5600 μg·h/mL about 5650 μg·h/mL, about 5700 μg·h/mL about 5750 μg·h/mL about 5800 μg·h/mL about 5850 μg·h/mL, about 5900 μg·h/mL about 5950 μg·h/mL about 6000 μg·h/mL about 6050 μg·h/mL, about 6100 μg·h/mL about 6150 μg·h/mL about 6200 μg·h/mL, about 6250 μg·h/mL, about 6300 μg·h/mL about 6350 μg·h/mL about 6400 μg·h/mL about 6450 μg·h/mL, about 6500 μg·h/mL about 6550 μg·h/mL about 6600 μg·h/mL about 6650 μg·h/mL, about 6700 μg·h/mL about 6750 μg·h/mL about 6800 μg·h/mL about 6850 μg·h/mL/mL, about 6900 μg·h/mL, about 6950 μg·h/mL, or about 7000 μg·h/mL.

In one embodiment, a pharmaceutical composition of the present disclosure provides a mean total eicosapentaenoic acid, unesterified plasma eicosapentaenoic acid, or total eicosapentaenoic acid red blood cell C_(min) of about 0.2 μg/mL to about 250 μg/mL, for example about 0.2 μg/mL to about 2 μg/mL, about 20 μg/mL to about 70 μg/mL, about 70 μg/mL to about 215 μg/mL, about 0.2 μg/mL, about 0.3 μg/mL, about 0.4 μg/mL (e.g., about 0.36 μg/mL, about 0.41 μg/mL or about 0.44 μg/mL), about 0.5 μg/mL, about 0.6 μg/mL, about 0.7 μg/mL, about 0.8 μg/mL, about 0.9 μg/mL, about 1 μg/mL (e.g., about 1.06 μg/mL), about 1.1 μg/mL, about 1.2 μg/mL, about 1.3 μg/mL, about 1.4 μg/mL, about 1.5 μg/mL, about 1.6 μg/mL, about 1.7 μg/mL, about 1.8 μg/mL, about 1.9 μg/mL, about 2 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 18 μg/mL, about 20 μg/mL, about 21 μg/mL (e.g., about 20.7 μg/mL), about 25 μg/mL, about 26 μg/mL (e.g., about 26.3 μg/mL), about 30 μg/mL, about 31 μg/mL (e.g., about 31.2 μg/mL), about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 64 μg/mL (e.g., about 64.1 μg/mL), about 65 μg/mL, about 70 μg/mL (e.g., about 69.7 μg/mL), about 75 μg/mL (e.g., about 75.1 μg/mL), about 80 μg/mL, about 84 μg/mL (e.g., about 83.5 μg/mL), about 85 μg/mL, about 89 μg/mL (e.g., about 89.3 μg/mL), about 90 μg/mL, about 95 μg/mL, about 100 μg/mL, about 101 μg/mL, about 104 μg/mL, about 105 μg/mL, about 110 μg/mL, about 115 μg/mL, about 120 μg/mL, about 125 μg/mL, about 130 μg/mL, about 135 μg/mL, about 140 μg/mL, about 145 μg/mL, about 150 μg/mL, about 155 μg/mL, about 160 μg/mL, about 165 μg/mL, about 170 μg/mL, about 175 μg/mL, about 180 μg/mL, about 185 μg/mL, about 190 μg/mL, about 195 μg/mL, about 200 μg/mL, about 205 μg/mL, about 210 μg/mL, about 215 μg/mL, about 220 μg/mL, about 225 μg/mL, about 230 μg/mL, about 235 μg/mL, about 240 μg/mL, about 245 μg/mL, or about 250 μg/mL.

In one embodiment, a pharmaceutical composition of the present disclosure provides a mean total eicosapentaenoic acid, unesterified plasma eicosapentaenoic acid, or total eicosapentaenoic acid red blood cell T_(max) of about 1 hour to about 36 hours, for example about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours.

In one embodiment, a pharmaceutical composition of the present disclosure provides a mean total eicosapentaenoic acid, unesterified plasma eicosapentaenoic acid, or total eicosapentaenoic acid red blood cell T_(1/2) of about 50 hours to about 700 hours, for example about 50 hours, about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 75 hours, about 80 hours, about 85 hours, about 90 hours, about 95 hours, about 100 hours, about 105 hours, about 110 hours, about 115 hours, about 120 hours, about 125 hours, about 130 hours, about 135 hours, about 140 hours, about 145 hours, about 150 hours, about 155 hours, about 160 hours, about 165 hours, about 170 hours, about 175 hours, about 180 hours, about 185 hours, about 190 hours, about 195 hours, about 200 hours, about 205 hours, about 210 hours, about 215 hours, about 220 hours, about 225 hours, about 230 hours, about 235 hours, about 240 hours, about 245 hours, about 250 hours, about 255 hours, about 260 hours, about 265 hours, about 270 hours, about 275 hours, about 280 hours, about 285 hours, about 290 hours, about 295 hours, about 300 hours, about 305 hours, about 310 hours, about 315 hours, about 320 hours, about 325 hours, about 330 hours, about 335 hours, about 340 hours, about 345 hours, about 350 hours, about 355 hours, about 360 hours, about 365 hours, about 370 hours, about 375 hours, about 380 hours, about 385 hours, about 390 hours, about 395 hours, about 400 hours, about 405 hours, about 410 hours, about 415 hours, about 420 hours, about 425 hours, about 430 hours, about 435 hours, about 440 hours, about 445 hours, about 450 hours, about 455 hours, about 460 hours, about 465 hours, about 470 hours, about 475 hours, about 480 hours, about 485 hours, about 490 hours, about 495 hours, about 500 hours, about 505 hours, about 510 hours, about 515 hours, about 520 hours, about 525 hours, about 530 hours, about 535 hours, about 540 hours, about 545 hours, about 550 hours, about 555 hours, about 560 hours, about 565 hours, about 570 hours, about 575 hours, about 580 hours, about 585 hours, about 590 hours, about 595 hours, about 600 hours, about 605 hours, about 610 hours, about 615 hours, about 620 hours, about 625 hours, about 630 hours, about 635 hours, about 640 hours, about 645 hours, about 650 hours, about 655 hours, about 660 hours, about 665 hours, about 670 hours, about 675 hours, about 680 hours, about 685 hours, about 690 hours, about 695 hours, or about 700 hours.

In one embodiment, a pharmaceutical composition of the present disclosure provides a mean total eicosapentaenoic acid, unesterified plasma eicosapentaenoic acid, or total eicosapentaenoic acid red blood cell CL/F of about 500 mL/hr to about 400,000 mL/hr, for example about 500 mL/hr, about 525 mL/hr, about 550 mL/hr, about 575 mL/hr, about 600 mL/hr, about 625 mL/hr, about 650 mL/hr, about 675 mL/hr, about 684 mL/hr (e.g., about 683.7 mL/hr), about 658 mL/hr (e.g., about 684.5 mL/hr), about 700 mL/hr, about 725 mL/hr, about 750 mL/hr, about 775 mL/hr, about 776 mL/hr (e.g., about 776.4 mL/hr), about 800 mL/hr, about 825 mL/hr, about 850 mL/hr, about 868 mL/hr (e.g., about 867.7 mL/hr), about 875 mL/hr, about 900 mL/hr, about 925 mL/hr, about 950 mL/hr, about 975 mL/hr, about 1,000 mL/hr, about 200,000 mL/hr, about 210,000 mL/hr, about 220,000 mL/hr, about 230,000 mL/hr, about 240,000 mL/hr, about 250,000 mL/hr, about 260,000 mL/hr, about 270,000 mL/hr, about 280,000 mL/hr, about 290,000 mL/hr, about 300,000 mL/hr, about 310,000 mL/hr, about 320,000 mL/hr, about 330,000 mL/hr, about 340,000 mL/hr, about 350,000 mL/hr, about 360,000 mL/hr, about 370,000 mL/hr, about 380,000 mL/hr, about 390,000 mL/hr, or about 400,000 mL/hr.

In one embodiment, a pharmaceutical composition of the present disclosure provides a mean total eicosapentaenoic acid, unesterified plasma eicosapentaenoic acid, or total eicosapentaenoic acid red blood cell V_(z)/F of about 50 L to about 50,000 L, for example about 75 L to about 90 L, about 28,000 L to about 41,000 L, about 50 L, about 55 L, about 60 L, about 65 L, about 70 L, about 75 L, about 79 L (e.g., about 79.3 L), about 80 L (e.g., about 79.8 L), about 85 L, about 88 L (e.g., about 88.4 L), about 90 L, about 95 L, about 100 L, about 28,000 L, about 28,200 L, about 29,000 L, about 30,000 L, about 31,000 L, about 32,000 L, about 33,000 L, about 33,160 L (e.g., about 33,164 L), about 34,000 L, about 35,000 L, about 36,000 L, about 37,000 L, about 38,000 L, about 39,000 L, about 40,000 L, about 40,870 L (e.g., about 40,873 L), about 41,000 L, about 42,000 L, about 43,000 L, about 44,000 L, about 45,000 L, about 46,000 L, about 47,000 L, about 48,000 L, about 49,000 L, or about 50,000 L.

In one embodiment, a pharmaceutical composition of the present disclosure comprising about 1 g of ethyl eicosapentaenoate (e.g., about 800 mg to about 1200 mg, about 850 mg to about 1150 mg, about 900 mg to about 1100 mg, about 950 mg to about 1050 mg, or about 975 mg to about 1025 mg) provides a mean plasma C_(max) of about 154.9+/−49.4 μg/mL, a mean plasma AUC_(0-24h) of about 2907+/−1160 μg·h/mL, a median plasma T_(max) of about 5 hours, and a mean plasma T_(1/2) of about 75.1+/−46.5 hours of ethyl eicosapentaenoate when administered orally twice per day to a human subject. In some embodiments, the pharmaceutical composition further provides a mean plasma C_(min) of about 101.0+/−50.4 μg/mL of ethyl eicosapentaenoate. In some embodiments, the preceding claims, wherein the pharmaceutical composition further provides a mean plasma CL/F of about 776.4+/−256.9 mL/h of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition further provides a mean plasma V_(z)/F of about 79.8+/−62.6 L of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition provides a mean RBC C_(max) of about 42.3+/−14.0 μg/mL, a mean RBC AUC_(0-24h) of about 801.5+/−268.5 μg·h/mL, a median RBC T_(max) of about 12 hours, and a mean RBC T_(1/2) of about 683.8 hours of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition further provides a mean RBC C_(min) of about 31.2+/−12.2 μg/mL of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition further provides a mean plasma C_(max) of about 0.66+/−0.34 μg/mL, a mean plasma AUC_(0-24h) of about 6.9+/−3.1 μg·h/mL, a median plasma T_(max) of about 5 hours, and a mean plasma T_(1/2) of about 80.8+/−37.5 hours of unesterified eicosapentaenoic acid when administered orally twice per day to a human subject. In some embodiments, the pharmaceutical composition further provides a mean plasma C_(min) of about 0.41+/−0.28 μg/mL of unesterified eicosapentaenoic acid. In some embodiments, the pharmaceutical composition further provides a mean plasma CL/F of about 364,513+/−214,003 mL/h of unesterified eicosapentaenoic acid. In some embodiments, the pharmaceutical composition further provides a mean plasma V_(z)/F of about 33,164+/−8,068 L of unesterified eicosapentaenoic acid.

In some embodiments, a pharmaceutical composition comprising about 2 g of ethyl eicosapentaenoate (e.g., about 1600 mg to about 2400 mg, about 1650 mg to about 2350 mg, about 1700 mg to about 2300 mg, about 1750 mg to about 2250 mg, about 1800 mg to about 2200 mg, about 1850 mg to about 2150 mg, about 1900 mg to about 2100 mg, or about 1950 mg to about 2050 mg) provides a mean plasma C_(max) of about 347.2+/−112.5 μg/mL, a mean plasma AUC_(0-24h) of about 6519+/−1963 μg·h/mL, a median plasma T_(max) of about 5 hours, and a mean plasma T_(1/2) of about 89.3+/−42.0 hours of ethyl eicosapentaenoate when administered orally twice per day to a human subject. In some embodiments, the pharmaceutical composition further provides a mean plasma C_(min) of about 101.0+/−50.4 μg/mL of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition further provides a mean plasma CL/F of about 683.7+/−280.6 mL/h of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition further provides a mean plasma V_(z)/F of about 88.4+/−55.2 L of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition further provides a mean RBC C_(max) of about 76.7+/−25.2 μg/mL, a mean RBC AUC_(0-24h) of about 1472+/−469.5 μg·h/mL, a median RBC T_(max) of about 8 hours, and a mean RBC T_(1/2) of about 371.4+/−311.5 hours of ethyl eicosapentaenoate. In some embodiments, the pharmaceutical composition further provides a mean RBC C_(min) of about 64.1+/−21.8 μg/mL of ethyl eicosapentaenoate.

In some embodiments, the pharmaceutical composition provides a mean plasma C_(max) of about 1.4+/−0.41 μg/mL, a mean plasma AUC_(0-24h) of about 18.4+/−4.6 μg·h/mL, a median plasma T_(max) of about 5 hours, and a mean plasma T_(1/2) of about 97.2+/−36.5 hours of unesterified eicosapentaenoic acid. In some embodiments, the pharmaceutical composition further provides a mean plasma C_(min) of about 1.06+/−0.56 μg/mL of unesterified eicosapentaenoic acid. In some embodiments, the pharmaceutical composition further provides a mean plasma CL/F of about 234,329+/−81,639 mL/h of unesterified eicosapentaenoic acid. In some embodiments, the pharmaceutical composition further provides a mean plasma V_(z)/F of about 28,200+/−10,929 L of unesterified eicosapentaenoic acid.

In some embodiments, the pharmaceutical composition is administered in unit dose form, wherein each unit dose includes about 500 mg or about 1000 mg of ethyl eicosapentaenoate. In some embodiments, unit dose forms are administered about 1 to about 4 times per day, for example about 1 time per day, about 2 times per day, about 3 times per day, or about 4 times per day.

EXAMPLES Example 1. Ethyl Eicosapentaenoic Acid in Subjects Having Fasting Triglycerides of About 200 mg/dL to Less Than 500 mg/dL

A multi-center, placebo-controlled, randomized, double-blind, 12-week study was performed to evaluate the efficacy and safety of >96% E-EPA in patients with fasting triglyceride levels ≥200 mg/dl and <500 mg/dl despite statin therapy (the mean of two qualifying entry values needed to be ≥185 mg/dl and at least one of the values needed to be ≥200 mg/dl). The primary objective of the study was to determine the efficacy of >96% E-EPA 2 g daily and 4 g daily, compared to placebo, in lowering fasting TG levels in patients with high risk for cardiovascular disease and with fasting TG levels 200 mg/dL and <500 mg/dL, despite treatment to LDL-C goal on statin therapy.

The secondary objectives of this study were the following:

-   1. To determine the safety and tolerability of >96% E-EPA 2 g daily     and 4 g daily; -   2. To determine the effect of >96% E-EPA on lipid and apolipoprotein     profiles including total cholesterol (TC), non-high-density     lipoprotein cholesterol (non-HDL-C), low density lipoprotein     cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C),     and very high density lipoprotein cholesterol (VHDL-C); -   3. To determine the effect of >96% E-EPA (on lipoprotein associated     phospholipase A₂ (Lp-PLA₂) from baseline to week 12; -   4. To determine the effect of >96% E-EPA on low-density lipoprotein     (LDL) particle number and size; -   5. To determine the effect of >96% E-EPA on oxidized LDL; -   6. To determine the effect of >96% E-EPA on fasting plasma glucose     (FPG) and hemoglobin A_(1c) (HbA_(1c)); -   7. To determine the effect of >96% E-EPA on insulin resistance; -   8. To determine the effect of >96% E-EPA on high-sensitivity     C-reactive protein (hsCRP); -   9. To determine the effects of >96% E-EPA 2 g daily and 4 g daily on     the incorporation of fatty acids into red blood cell membranes and     into plasma phospholipids; -   10. To explore the relationship between baseline fasting TG levels     and the reduction in fasting TG levels; and -   11. To explore the relationship between changes of fatty acid     concentrations in plasma and red blood cell membranes, and the     reduction in fasting TG levels.

The population for this study was men and women >18 years of age with a body mass index ≤45 kg/m² with fasting TG levels greater than or equal to 200 mg/dl and less than 500 mg/dl and on a stable does of statin therapy (with or without ezetimibe). The statin was atorvostatin, rosuvastatin or simvastatin. The dose of statin must have been stable for ≥4 weeks prior to the LDL-C/TG baseline qualifying measurement for randomization. The statin dose was optimized such that the patients are at their LDL-C goal at the LDL-C/TG baseline qualifying measurements. The same statin at the same dose was continued until the study ended.

Patients taking any additional non-statin, lipid-altering medications (niacin >200 mg/day, fibrates, fish oil, other products containing omega-3 fatty acids, or other herbal products or dietary supplements with potential lipid-altering effects), either alone or in combination with statin therapy (with or without ezetimibe), must have been able to safely discontinue non-statin, lipid-altering therapy at screening.

Patients at high risk for CVD, i.e., patients with clinical coronary heart disease (CHD) or clinical CHD risk equivalents (10-year risk >20%) as defined in the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) Guidelines were eligible to participate in this study. Those included patients with any of the following criteria: (1) Known CVD, either clinical coronary heart disease (CHD), symptomatic carotid artery disease (CAD), peripheral artery disease (PAD) or abdominal aortic aneurism; or (2) Diabetes Mellitus (Type 1 or 2).

Approximately 702 patients were randomized at approximately 80 centers in the U.S. The study was a 18- to 20-week, Phase 3, multi-center study consisting of 2 study periods: (1) A 6- to 8-week screening period that included a diet and lifestyle stabilization, a non-statin lipid-altering treatment washout, and an LDL-C and TG qualifying period and (2) A 12-week, double-blind, randomized, placebo-controlled treatment period.

During the screening period and double-blind treatment period, all visits were within ±3 days of the scheduled time. All patients continued to take the statin product (with or without ezetimibe) at the same dose they were taking at screening throughout their participation in the study.

The 6- to 8-week screening period included a diet and lifestyle stabilization, a non-statin lipid-altering treatment washout, and an LDL-C and TG qualifying period. The screening visit (Visit 1) occurred for all patients at either 6 weeks (for patients on stable statin therapy [with or without ezetimibe] at screening) or 8 weeks (for patients who will require washout of their current non-statin lipid-altering therapy at screening) before randomization, as follows:

-   -   Patients who did not require a washout: The screening visit         occurred at Visit 1 (Week −6). Eligible patients entered a         4-week diet and lifestyle stabilization period. At the screening         visit, all patients received counseling regarding the importance         of the National Cholesterol Education Program (NCEP) Therapeutic         Lifestyle Changes (TLC) diet and received basic instructions on         how to follow this diet.     -   Patients who required a washout: The screening visit occurred at         Visit 1 (Week −8). Eligible patients began a 6-week washout         period at the screening visit (i.e. 6 weeks washout before the         first LDL-C/TG qualifying visit). Patients received counseling         regarding the NCEP TLC diet and received basic instructions on         how to follow this diet. Site personnel contacted patients who         did not qualify for participation based on screening laboratory         test results to instruct them to resume their prior         lipid-altering medications.

At the end of the 4-week diet and lifestyle stabilization period or the 6-week diet and stabilization and washout period, eligible patients entered the 2-week LDL-C and TG qualifying period and had their fasting LDL-C and TG levels measured at Visit 2 (Week −2) and Visit 3 (Week −1). Eligible patients must have had an average fasting LDL-C level ≥40 mg/dL and <100 mg/dL and an average fasting TG level 200 mg/dL and <500 mg/dL to enter the 12-week double-blind treatment period. The LDL-C and TG levels for qualification were based on the average (arithmetic mean) of the Visit 2 (Week −2) and Visit 3 (Week −1) values. If a patient's average LDL-C and/or TG levels from Visit 2 and Visit 3 fell outside the required range for entry into the study, an additional fasting lipid profile was collected 1 week later at Visit 3.1. If a third sample was collected at Visit 3.1, entry into the study was based on the average (arithmetic mean) of the values from Visit 3 and Visit 3.1.

After confirmation of qualifying fasting LDL-C and TG values, eligible patients entered a 12-week, randomized, double-blind treatment period. At Visit 4 (Week 0), patients were randomly assigned to 1 of the following treatment groups:

-   -   >96% E-EPA 2 g daily,     -   >96% E-EPA 4 g daily, or     -   Placebo.

Approximately 216 patients per treatment group were randomized in this study. Stratification was by type of statin (atorvastatin, rosuvastatin or simvastatin), the presence of diabetes, and gender. Baseline parameters for randomized subjects in this study are shown in Table 1 below. The lower-efficacy statin regimen included administration of 5-10 mg of simvastatin per day; the medium-efficacy statin regimens included administration of 5-10 mg rosuvastatin per day, 10-20 mg of atorvastatin per day, 20-40 mg of simvastatin per day, or 10-20 mg of simvastatin and 5-10 mg of ezetimibe per day; and the higher-efficacy statin regimens included administration of 20-40 mg of rosuvastatin per day, 40-80 mg of atorvastatin per day, 80 mg of simvastatin per day, or 40-80 mg of simvastatin and 5-10 mg of ezetimibe per day.

TABLE 1 Patient Characteristics (Randomized Population) AMR101 AMR101 4 g/day 2 g/day Placebo (n = 233) (n = 236) (n = 233) Age, y, mean (SD) 61.1 (10.03) 61.8 (9.42) 61.2 (10.05) Male, n (%) 142 (60.9) 144 (61.0) 145 (62.2) White, n (%) 226 (97.0) 226 (95.8) 224 (96.1) Weight, kg, 94.5 (18.30) 95.5 (18.29) 97.0 (19.14) mean (SD) BMI, kg/m², 32.7 (4.99) 32.9 (4.98) 33.0 (5.04) mean (SD) Diabetes, n (%) 171 (73.4) 172 (72.9) 171 (73.4) Baseline TG >750 NA NA NA mg/dL, n (%) Statin use, n (%): Any 233 (100) 236 (100) 233 (100) Atorvastatin 44 (18.9) 43 (18.2) 45 (19.3) Simvastatin 134 (57.5) 136 (57.6) 133 (57.1) Rosuvastatin 55 (23.6) 57 (24.2) 55 (23.6) Statin efficacy regimens, n (%) Lower 16 (6.9) 17 (7.2) 15 (6.4) Medium 148 (63.5) 148 (62.7) 144 (61.8) Higher 69 (29.6) 71 (30.1) 74 (31.8)

Baseline lipid parameters for subjects in the intent-to-treat (“ITT”) population of this study are shown in Table 2 below. Data are presented as median (interquartile range) values.

TABLE 2 Baseline Lipid Parameters (ITT Population) AMR101 AMR101 4 g/day 2 g/day Placebo Lipid Parameter (IQR) (IQR) (IQR) (mg/dL) (n = 233) (n = 236) (n = 233) TG 264.8 (93.0) 254.0 (92.5) 259.0 (81.0) (n = 226) (n = 234) (n = 227) LDL-C  82.0 (25.0)  82.0 (24.0)  84.0 (27.0) (n = 225) (n = 233) (n = 226) Non-HDL-C 128.0 (32.0) 128.0 (33.0) 128.0 (34.0) (n = 226) (n = 234) (n = 227) TC 167.0 (38.0) 169.0 (34.0) 168.0 (38.0) (n = 226) (n = 234) (n = 227) HDL-C  37.0 (12.0)  38.0 (13.0)  39.0 (12.0) (n = 226) (n = 234) (n = 227)

During the double-blind treatment period, patients returned to the site at Visit 5 (Week 4), Visit 6 (Week 11), and Visit 7 (Week 12) for efficacy and safety evaluations.

Eligible patients were randomly assigned at Visit 4 (Week 0) to receive orally >96% E-EPA 2 g daily, >96% E-EPA 4 g daily, or placebo.

>96% E-EPA was provided in 1 g liquid-filled, oblong, gelatin capsules. The matching placebo capsule was filled with light liquid paraffin and contained 0 g of >96% E-EPA. >96% E-EPA capsules were to be taken with food (i.e. with or at the end of a meal).

During the double-blind treatment period, patients were to take 2 capsules (>96% E-EPA or matching placebo) in the morning and 2 capsules in the evening for a total of 4 capsules per day.

-   -   Patients in the >96% E-EPA 2 g/day treatment group received         1 >96% E-EPA 1 g capsule and 1 matching placebo capsule in the         morning and in the evening.     -   Patients in the >96% E-EPA 4 g/day treatment group received         2 >96% E-EPA 1 g capsules in the morning and evening.     -   Patients in the placebo group received 2 matching placebo         capsules in the morning and evening.

The primary efficacy variable for the double-blind treatment period was percent change in TG from baseline to Week 12 endpoint. The secondary efficacy variables for the double-blind treatment period included the following:

-   -   Percent changes in total cholesterol (TC), high-density         lipoprotein cholesterol (HDL-C), LDL-C, calculated non-HDL-C,         and very low-density lipoprotein cholesterol (VLDL-C) from         baseline to Week 12 endpoint;     -   Percent change in very low-density lipoprotein TG from baseline         to Week 12;     -   Percent changes in apolipoprotein A-I (apo A-I), apolipoprotein         B (apo B), and apo A-I/apo B ratio from baseline to Week 12;     -   Percent changes in lipoprotein(a) from baseline to Week 12;     -   Percent changes in LDL particle number and size, measured by         nuclear magnetic resonance, from baseline to Week 12;     -   Percent change in remnant-like particle cholesterol from         baseline to Week 12;     -   Percent change in oxidized LDL from baseline to Week 12;     -   Changes in FPG and HbA_(1c) from baseline to Week 12;     -   Change in insulin resistance, as assessed by the homeostasis         model index insulin resistance, from baseline to Week 12;     -   Percent change in lipoprotein associated phospholipase A₂         (Lp-PLA₂) from baseline to Week 12;     -   Change in intracellular adhesion molecule-1 from baseline to         Week 12;     -   Change in interleukin-2 from baseline to Week 12;     -   Change in plasminogen activator inhibitor-1 from baseline to         Week 12. Note: this parameter will only be collected at sites         with proper storage conditions;     -   Change in hsCRP from baseline to Week 12; and     -   Change in plasma concentration and red blood cell membrane         content of fatty acid from baseline to Week 12 including EPA,         docosapentaenoic acid (DPA), docosahexaenoic acid (DHA),         arachidonic acid (AA), dihomo-γ-linolenic acid (DGLA), the ratio         of EPA/AA, ratio of oleic acid/stearic acid (OA/SA), and the         ratio of total omega-3 acids over total omega-6 acids.

Safety assessments included adverse events, clinical laboratory measurements (chemistry, hematology, and urinalysis), 12-lead electrocardiograms (ECGs), vital signs, and physical examinations.

For TG, TC, HDL-C, LDL-C, calculated non-HDL-C, and VLDL-C, baseline was defined as the average of Visit 4 (Week 0) and the preceding lipid qualifying visit (either Visit 3 [Week −1] or if it occurs, Visit 3.1) measurements. Baseline for all other efficacy parameters was the Visit 4 (Week 0) measurement.

For TG, TC, HDL-C, LDL-C, calculated non-HDL-C, and VLDL-C, Week 12 endpoint was defined as the average of Visit 6 (Week 11) and Visit 7 (Week 12) measurements.

Week 12 endpoint for all other efficacy parameters were the Visit 7 (Week 12) measurement.

The primary efficacy analysis was performed using a 2-way analysis of covariance (ANCOVA) model with treatment as a factor and baseline TG value as a covariate. The least-squares mean, standard error, and 2-tailed 95% confidence interval for each treatment group and for each comparison were estimated. The same 2-way ANCOVA model was used for the analysis of secondary efficacy variables.

The primary analysis was repeated for the per-protocol population to confirm the robustness of the results for the intent-to-treat population.

Non-inferiority tests for percent change from baseline in LDL-C were performed between >96% E-EPA doses and placebo using a non-inferiority margin of 6% and a significant level at 0.05.

For the following key secondary efficacy parameters, treatment groups were compared using Dunnett's test to control the Type 1 error rate: TC, LDL-C, HDL-C, non-HDL-C, VLDL-C, Lp-PLA₂, and apo B. For the remaining secondary efficacy parameters, Dunnett's test was be used and the ANCOVA output were considered descriptive.

The evaluation of safety was based primarily on the frequency of adverse events, clinical laboratory assessments, vital signs, and 12-lead ECGs. The primary efficacy variable is the percent change in fasting TG levels from baseline to Week 12. A sample size of 194 completed patients per treatment group provided 90.6% power to detect a difference of 15% between >96% E-EPA and placebo in percent change from baseline in fasting TG levels, assuming a standard deviation of 45% in TG measurements and a significance level of p<0.05.

Previous data on fasting LDL-C show a difference in percent change from baseline of 2.2%, with a standard deviation of 15%, between study drug and placebo. A sample size of 194 completed patients per treatment group provided 80% power to demonstrate non-inferiority (p<0.05, one-sided) of the LDL-C response between >96% E-EPA 4 g daily and placebo, within a 6% margin. To accommodate a 10% drop-out rate from randomization to completion of the double-blind treatment period, a total of 648 randomized patients was planned (216 patients per treatment group); 702 subjects were randomized, as further described below.

A summary of baseline values, end-of-treatment values, and median placebo-adjusted percent change from baseline to study end of inflammation-associated end points in each of the three ITT sub-populations is shown in Table 3.

TABLE 3 Median Placebo-adjusted Percent Change From Baseline to Study End in Inflammation-associated End Points (ITT Populations). AMR101 4 g/day AMR101 2 g/day Placebo Median Placebo-adjusted % End-of- Change End-of- Change End-of- Change Change From Baseline Baseline treatment from Baseline treatment from Baseline treatment from AMR101 AMR101 value value baseline, value value baseline, value value baseline, 4 g/day vs 2 g/day vs (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR) placebo, P placebo, P MARINE n = 76 n = 73 n = 75 ICAM-1 (ng/mL) 250.0 253.0  −0.9 255.5 257.5 −0.4 247.5 246.0  2.6 −2.5 −2.3 (n = 75, 70, 72) (85.00) (89.00) (10.69) (86.00) (102.00) (12.29) (101.00) (89.50) (13.43) 0.1188 0.2201 Ox-LDL (U/L)  78.8  74.8  −3.0  76.2  75.2  0.4  74.3  74.8  3.3 −6.4 −3.0 (n = 74, 70, 71) (26.51) (26.52) (2.35) (24.00) (23.52) (2.41) (24.86) (23.32) (2.39) 0.0599 0.3818 Lp-PLA₂ (ng/mL) 246.0 201.0 −17.1 235.0 220.5 −5.1 253.0 256.0 −2.4 −13.6 −5.1 (n = 73, 70, 70) (116.00) (100.00) (24.43) (106.00) (101.00) (24.14) (126.00) (146.00) (29.35) 0.0003 0.1529 IL-6 (pg/mL)  2.3  2.4    0.3  3.0  3.0  3.4  2.5  2.3  5.3 11.0 4.7 (n = 60, 62, 61) (3.34) (3.32) (93.07) (2.78) (5.39) (76.70) (4.12) (1.85) (79.27) 0.3629 0.6654 hsCRP (mg/L)  2.2  2.2  −2.5  2.0  2.4 25.1  1.8  2.5 33.3 −36.0 −10.1 (n = 75, 70, 72) (3.10) (2.90) (81.19) (2.70) (3.20) (96.43) (3.05) (3.95) (80.49) 0.0012 0.4028 ANCHOR n = 226 n = 234 n = 227 ICAM-1 (ng/mL) 273.0 270.0    0.8 267.0 268.5  0.5 269.0 257.0  3.6 −2.4 −2.2 (n = 78, 74, 83) (96.00) (110.00) (13.27) (97.00) (89.00) (12.41) (122.00) (131.00) (12.07) 0.1888 0.1944 Ox-LDL (U/L)  54.0  51.4  −4.8  54.0  55.8  2.6  51.8  59.7 11.6 −13.3 −5.8 (n = 78, 75, 84) (14.60) (17.50) (19.63) (17.80) (22.80) (18.28) (16.80) (18.10) (28.09) <0.0001 0.0946 Lp-PLA₂ (ng/mL) 180.0 160.0 −12.8 190.0 183.5 −1.8 185.0 200.0  6.7 −19.0 −8.0 (n = 217, 224, 213) (56.00) (57.00) (18.52) (55.50) (57.50) (23.11) (58.00) (71.00) (24.03) <0.0001 <0.0001 IL-6 (pg/mL)  2.7  2.6   3.1  2.4  2.7  6.9  3.2  2.9  3.3 −1.0 7.0 (n = 78, 74, 83) (2.61) (2.08) (56.47) (2.01) (2.28) (51.85) (3.23) (2.95) (59.37) 0.9031 0.3643 hsCRP (mg/L)  2.2  2.0  −2.4  1.9  2.5 10.3  2.2  2.6 17.1 −22.0 −6.8 (n = 217, 227, 219) (2.70) (3.00) (62.75) (2.90) (3.40) (88.61) (4.00) (4.70) (107.99) 0.0005 0.2889

Only subsets with non-missing baseline and week 12 values are included in the data of Table 3. Data are presented as median (interquartile range) for end point values. End Point Abbreviations: hsCRP, high-sensitivity C-reactive protein; ICAM-1, intracellular adhesion molecule-1; IL-6, interleukin-6; IQR, interquartile range; Lp-PLA2, lipoprotein-associated phospholipase A2; Ox-LDL, oxidized low-density lipoprotein.

Results

Of the 702 randomized subjects, 687 were in the intent-to-treat (“ITT”) population as follows:

-   -   Ultra-pure EPA, 4 g/day: 226 subjects     -   Ultra-pure EPA, 2 g/day: 234 subjects     -   Placebo: 227 subjects

Lipids were extracted from plasma and red blood cell (“RBC”) suspensions and converted into fatty acid methyl esters for analysis using a standard validated gas chromatography/flame ionization detection method. Fatty acid parameters were compared between EPA treatment groups and placebo using an ANCOVA model with treatment, gender, type of statin therapy, and presence of diabetes as factors, and the baseline parameter value as a covariate. LSMs, SEs, and 2-tailed 95% confidence intervals for each treatment group and for each comparison were determined.

Baseline characteristics of the three ITT groups were comparable, with 61.4% of the ITT subjects being male, 96.3% being white, having a mean age of 61.4 years, a weight of 95.7 kg and a BMI of 32.9 kg/m². ITT subjects with incomplete fatty acid data at baseline and/or at 12 weeks were excluded from the analyses described below (as reflected in the n numbers shown in Tables 4-6).

As shown in Table 4, ultra-pure EPA at 2 g/day and 4 g/day both significantly increased mean placebo-adjusted plasma concentrations of EPA, DPAn-3, and the ratio of total omega-3 to omega-6 fatty acids (“total ω-3/total ω-6”). In addition, both 2 g/day and 4 g/day doses of ultra-pure EPA significantly decreased mean placebo-adjusted plasma concentrations of arachidonic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and the plasma ratio of arachidonic acid to EPA. Neither dose of ultra-pure EPA produced a significant change in mean placebo-adjusted plasma concentration of DHA.

TABLE 4 Change from Baseline to Week 12 in Plasma Fatty Acid Concentrations and Ratios. Placebo-adjusted percent change from IPE 4 g/day (n = 226) IPE 2 g/day (n = 234) Placebo (n = 227) baseline Change Change Change IPE IPE End-of- from End-of- from End-of- from 4 g/day vs 2 g/day vs Baseline treatment baseline Baseline treatment baseline Baseline treatment baseline placebo, placebo, (SD) (SD) (SE) (SD) (SD) (SE) (SD) (SD) (SE) %, P %, P EPA 19.7 147.7 137.7  26.6 89.3   71.3 24.3 24.6 10.3 649.7 268.2 n = 41, 50, 51 (6.1) (53.8) (5.9) (13.1) (39.7) (5.3) (29.0) (25.0) (5.3) <0.0001 <0.0001 DHA 51.6  53.6  2.3 58.9 62.7    5.0 58.6 62.6  5.1 −2.9 −0.7 n = 47, 51, 52 (15.5) (13.7) (2.0) (18.9) (18.6) (1.8) (34.3) (33.5) (1.8) 0.4925 0.8711 DPAn-3 19.9  50.2 31.8 23.3 44.3   22.4 21.6 24.0  3.9 143.6 88.9 n = 48, 51, 51 (5.2) (14.3) (1.8) (6.6) (13.5) (1.7) (7.9) (9.4) (1.7) <0.0001 <0.0001 AA 281.4  222.8 −60.1   301.2  266.7  −31.1 307.6  328.4  26.0 −30.7 −19.1 n = 49, 51, 51 (71.0) (66.1) (6.4) (73.5) (61.2) (6.0) (75.1) (67.8) (6.2) <0.0001 <0.0001 Palmitic 964.7  843.8 −161    1066    1004    −50.1 1045    1109    59.9 −23.3 −12.1 n = 47, 49, 50 (215.5) (215.6) (45.4) (291.6) (321.2) (43.1) (298.3) (351.0) (43.2) <0.0001 0.0139 Stearic 277.3  253.4 −31.2   302.5  276.2  −19.6 293.7  306.3  14.2 −16.5 −12.6 n = 49, 46, 49 (55.1) (48.5) (8.2) (61.8) (52.8) (8.2) (61.2) (61.8) (8.1) <0.0001 0.0003 Oleic 970.0  782.5 −219    1029    972.1  −61.7 1018    1102    71.3 −30.1 −14.3 n = 46, 49, 50 (256.8) (210.9) (49.6) (271.3) (328.9) (46.9) (295.3) (400.2) (47.1) <0.0001 0.0089 Linoleic 1066    921.8 −152    1125    1082    −35.0 1099    1229    128.6 −24.8 −14.5 n = 49, 49, 51 (263.6) (202.4) (38.6) (253.0) (285.0) (37.2) (257.3) (350.8) (36.8) <0.0001 0.0003 Ratio of 16.3  1.6 −14.1   13.2  3.4 −11.2 16.8 16.4  0.5 −90.3 −75.4 AA/EPA (6.7) (0.8) (0.6) (4.2) (1.7) (0.5) (6.8) (6.5) (0.5) <0.0001 <0.0001 n = 46, 50, 49 Ratio of total  0.1  0.2  0.2  0.1  0.2    0.1  0.1  0.1  0.0 165.9 64.3 ω-3/total ω-6 (0.01) (0.06) (0.01) (0.02) (0.04) (0.01) (0.04) (0.04) (0.01) <0.0001 <0.0001 n= 46, 51, 50 Mean and SD are reported for baseline and end-of-treatment values; LS mean and SE are reported for changes from baseline and placebo-adjusted changes from baseline.

As shown in Table 5, ultra-pure EPA at 2 g/day and 4 g/day both significantly increased mean placebo-adjusted concentrations of EPA, DPAn-3, and the ratio of total omega-3 to omega-6 fatty acids (“total ω-3/total ω-6”) in RBCs. In addition, both 2 g/day and 4 g/day doses of ultra-pure EPA significantly decreased mean placebo-adjusted RBC concentrations of arachidonic acid and the RBC ratio of arachidonic acid to EPA. The RBC concentration of linoleic acid significantly decreased over placebo for subjects in the 4 g/day EPA group. Both doses of ultra-pure EPA produced a significant change in mean placebo-adjusted RBC concentration of DHA.

TABLE 5 Change from Baseline to Week 12 in RBC Fatty Acid Concentrations and Ratios. Placebo-adjusted percent change from IPE 4 g/day (n = 226) IPE 2 g/day (n = 234) Placebo (n = 227) baseline Change Change Change IPE IPE End-of- from End-of- from End-of- from 4 g/day vs 2 g/day vs Baseline treatment baseline Baseline treatment baseline Baseline treatment baseline placebo, placebo, (SD) (SD) (SE) (SD) (SD) (SE) (SD) (SD) (SE) %, P %, P EPA  6.1  43.2   39.4  8.2  28.4   21.1  7.2  6.5   0.8 619.2 290.4 n = 48, 49, 47 (2.3) (18.2) (1.8) (2.7) (8.5) (1.8) (3.3) (3.3) (1.8) <0.0001 <0.0001 DHA  44.0  39.0  −6.4  50.5  44.7  −5.5  47.5  45.4 −2.5 −6.6 −5.4 n = 43, 48, 46 (11.8) (8.4) (1.0) (13.2) (11.4) (0.9) (11.8) (11.8) (0.9_ 0.0107 0.0310 DHAn-3  28.3  61.9   33.6  31.6  52.2   21.2  29.9  28.9 −0.8 122.4 74.9 n = 45, 50, 45 (5.6) (12.6) (1.4) (5.0) (8.9) (1.3) (5.3) (4.7) (1.4) <0.0001 <0.0001 AA 184.7 153.1 −37.5 190.9 173.0 −20.0 190.3 195.1   1.9 −20.3 −11.6 n = 43, 48, 46 (37.2) (28.8) (3.6) (28.6) (25.7) (3.3) (25.4) (25.9) (3.4) <0.0001 <0.0001 Palmitic 304.0 301.2  −6.3 317.3 321.1    4.9 314.8 315.4   0.9 −2.7 0.5 n = 45, 49, 49 (37.3) (37.0) (4.4) (37.2) (41.4) (4.1) (39.1) (30.2) (4.1) 0.1091 0.7706 Stearic 170.6 170.5  −1.4 174.9 178.7    3.9 174.5 176.0   1.4 −1.3 1.6 n = 44, 50, 45 (19.0) (18.9) (2.5) (22.4) (22.9) (2.3) (14.6) (16.5) (2.4) 0.4773 0.3753 Oleic 194.4 188.8  −8.0 201.7 204.7    3.1 200.0 200.2 −0.4 −3.7 1.6 n = 44, 50, 44 (27.6) (24.1) (3.1) (30.0) (31.6) (2.8) (23.3) (21.9) (3.0) 0.0498 0.3599 Linoleic 147.1 132.7 −16.1 154.3 153.1  −1.3 152.3 154.8   1.9 −11.1 −2.1 n = 47, 49, 47 (32.2) (26.2) (3.8) (33.4) (37.6) (3.5) (23.7) (31.6) (3.6) 0.0003 0.4709 Ratio of  32.0  3.6 −26.8  26.1  6.4 −21.9  31.0  32.8   3.0 −98.5 −87.1 AA/EPA (10.5) (1.7) (0.8) (8.2) (2.0) (0.8) (11.2) (9.6) (0.8) <0.0001 <0.0001 n = 45, 50, 49 Ratio of total  0.2  0.4    0.2  0.2  0.3    0.1  0.2  0.2 −0.0 123.0 60.4 ω-3/total ω-6 (0.03) (0.11) (0.01) (0.05) (0.06) (0.01) (0.04) (0.04) (0.01) <0.0001 <0.0001 n = 47, 52, 49 Mean and SD are reported for baseline and end-of-treatment values; LS mean and SE are reported for changes from baseline and placebo-adjusted changes from baseline.

Table 6 shows changes from baseline and placebo-adjusted percent changes from baseline in the proportion (mol %) of fatty acid classes to total fatty acids in both plasma and in RBCs. The monounsaturated fatty acid group included myristoleic, palmitoleic, cis-vaccenic, oleic, gondoic/gadoleic, erucic and nervonic acids. The saturated fatty acid group included myristic, palmitic, stearic, arachidic, behenic and lignoceric acids. The “total ω-3” group included α-linolenic, stearidonic, eicosatrienoic, ω-3 arachidonic, eicosapentaenoic, ω-3 docosapentaenoic and docosahecaenoic acids. The “total ω-6” group included linoleic, γ-linolenic, eicosadienoic, dihomo-γ-linolenic, ω-6 arachidonic, adrenic, and ω-6 docosapentaenoic acids. The “total fatty acid” group represented the sum of the monounsaturated, saturated, total ω-3 and total ω-6 groups. Treatment with 2 g/day or 4 g/day ultra-pure EPA resulted in an increased proportion of ω-3 fatty acids in plasma and in RBCs compared to placebo, and a decreased proportion of ω-6 fatty acids in plasma and in RBCs compared to placebo. Treatment with 4 g/day ultra-pure EPA also resulted in a decreased proportion of monounsaturated fatty acids in plasma and RBCs compared to placebo.

TABLE 6 Placebo-Adjusted Percent Change in the Proportion of Fatty Acid Classes to Total Fatty Acids. Placebo-adjusted percent change from IPE 4 g/day (n = 226) IPE 2 g/day (n = 234) Placebo (n = 227) baseline Change Change Change IPE IPE End-of- from End-of- from End-of- from 4 g/day vs 2 g/day vs Baseline treatment baseline Baseline treatment baseline Baseline treatment baseline placebo, placebo, (SD) (SD) (SE) (SD) (SD) (SE) (SD) (SD) (SE) %, P %, P Plasma Saturated 33.7 33.6 −0.5 34.3 33.6 −0.7 34.2 33.6 −0.7 0.4 −0.2 n = 49, 52, 52 (2.5) (2.4) (0.3) (2.2) (2.2) (0.3) (2.5) (2.4) (0.3) 0.6923 0.8387 Monounsaturated 28.6 26.7 −2.0 28.5 28.0 −0.7 28.7 28.5 −0.3 −5.8 −1.4 n = 49, 52, 52 (2.8) (3.0) (0.4) (2,2) (2.8) (0.4) (2.6) (3.3) (0.4) 0.0028 0.4631 Total ω-6 34.7 32.6 −1.8 34.0 33.3 −0.8 34.0 34.7   0.7 −7.4 −4.6 n = 49, 52, 52 (3.9) (3.5) (0.5) (2.7) (3.4) (0.5) (3.4) (3.8) (0.5) 0.0002 0.0171 Total ω-3  2.9  7.1   4.4  3.2  5.1   2.1 3.1  3.1   0.3 142.2 60.3 n = 49, 52, 52 (0.5) (2.0) (0.2) (0.7) (1.2) (0.2) (1.3) (1.2) (0.2) <0.0001 <0.0001 RBCs Saturated 43.4 44.2   0.5 43.7 43.5 −0.3 43.8 42.9 −0.9 3.2 1.3 n = 49, 52, 52 (2.2) (3.0) (0.3) (3.0) (2.1) (0.3) (2.6) (1.7) (0.3) 0.0009 0.1589 Monounsaturated 20.5 19.8 −0.5 19.9 19.9 −0.3 19.9 20.1   0.0 −2.7 −0.7 n = 49, 52, 52 (2.2) (1.7) (0.2) (1.2) (1.1) (0.2) (1.6) (1.9) (0.2) 0.0419 0.5771 Total ω-6 30.8 25.8 −4.9 30.3 28.1 −2.4 30.6 31.5   0.8 −19.7 −11.3 n = 49, 52, 52 (2.0) (3.2) (0.4) (3.1) (2.1) (0.4) (3.1) (2.2) (0.4) <0.0001 <0.0001 Total ω-3  5.4 10.2   4.9  6.1  8.5   2.6  5.7  5.5 −0.0 89.1 50.0 n = 49, 52, 52 (1.1) (2.5) (0.2) (1.3) (1.5) (0.2) (1.2) (1.1) (0.2) <0.0001 <0.0001 Mean and SD are reported for baseline and end-of-treatment values; LS mean and SE are reported for changes from baseline and placebo-adjusted changes from baseline. Total = sum of ω-3, ω-6, monounsaturated, and saturated fatty acids. ω-3 fatty acids = sum of α-linolenic, stearidonic, eicosatrienoic, ω-3 arachidonic, eicosapentaenoic, ω-3 docosapentaenoic, and docosahexaenoic acids. ω-6 fatty acids = sum of linoleic, γ-linolenic, eicosadienoic, dihomo-γ-linolenic, ω-6 arachidonic, adrenic, and ω-6 docosapentaenoic acids. Monounsaturated fatty acids = myristoleic, palmitoleic, cis-vaccenic, oleic, gondoic/gadoleic, erucic, and nervonic acids. Saturated fatty acids = sum of myristic, palmitic, stearic, arachidic, behenic, and lignoceric acids.

FIG. 1 shows the placebo-adjusted least squares mean changes (P<0.0001) in EPA concentration from baseline to week 12 in plasma and RBCs. The error bars represent 95% confidence intervals. Treatment with 4 g/day ultra-pure EPA (referred to as “IPE” in the Figures) resulted in a 649.7% placebo-adjusted LS mean increase in plasma EPA over baseline, and a 619.2% placebo-adjusted LS mean increase in RBC EPA over baseline. Treatment with 2 g/day ultra-pure EPA resulted in a 268.2% placebo-adjusted LS mean increase in plasma EPA over baseline, and a 290.4% placebo-adjusted LS mean increase in RBC EPA over baseline.

FIG. 2 shows plasma (dark diamonds) and RBC (light diamonds) placebo-adjusted least square mean changes from baseline to week 12 for select individual fatty acids and ratios of arachidonic acid to EPA (“AA/EPA”) and total ω-3 fatty acids to total ω-6 fatty acids (“n-3/n-6”) upon treatment with 4 g/day ultra-pure EPA (left) or 2 g/day ultra-pure EPA (right). The values for each data point and corresponding P values are reported in Tables 4 and 5. Error bars represent 95% confidence intervals.

FIG. 3 depicts the EPA dose dependence of changes in EPA plasma and RBC concentration from baseline. Increased doses of ultra-pure EPA resulted in increased EPA concentration in both plasma and RBCs. EPA concentration in plasma (dark circles, γ-axis on left) increased 10.3 μg/g for the placebo group, 71.3 μg/g for the 2 g/day ultra-pure EPA group, and 137.7 μg/g for the 4 g/day ultra-pure EPA group. EPA concentration in RBCs (light circles, y-axis on right) increased 0.8 μg/g for the placebo group, 21.1 μg/g for the 2 g/day ultra-pure EPA group, and 39.4 μg/g for the 4 g/day ultra-pure EPA group. Error bars represent 95% confidence intervals for each data point.

FIG. 4 shows the relationship between plasma triglyceride lowering and changes in EPA plasma and RBC concentrations (a pharmacokinetic/pharmacodynamic relationship). Increased EPA concentrations in plasma (FIG. 4A) and RBCs (FIG. 4B) were accompanied by greater reductions in plasma triglyceride levels. Triglyceride concentrations are presented in FIG. 4 as median percent changes from baseline. Vertical error bars represent the first and third quartile of the IQR. EPA concentrations are presented in FIG. 4 as mean percent changes from baseline. Horizontal error bars represent 95% confidence intervals.

As shown in FIG. 14, a linear concentration-response pharmacodynamics relationship between EPA plasma levels and triglyceride reduction was also observed. Error bars represent 95% confidence intervals for both median triglyceride percent change from baseline (y-axis; vertical error bars) and mean EPA plasma concentration percent change from baseline (x-axis; horizontal error bars). This data is consistent with the observation in all studies to-date that a greater increase in EPA concentrations in both plasma and RBCs was observed for subjects administered about 4 g per day of EPA compared to those administered about 2 g per day. In addition, median percent reductions in triglycerides (compared to baseline) were higher for subjects administered about 4 grams of EPA per day than for those administered about 2 g of EPA per day in both the ANCHOR study (described herein and also in U.S. Pat. No. 8,410,486, incorporated herein by reference) and also in the MARINE study (see, e.g., U.S. Pat. No. 8,357,677, incorporated herein by reference).

These data indicate that ultra-pure EPA at either 2 g/day or 4 g/day significantly increased the concentrations of EPA and its metabolite, DPA n-3, in plasma and RBCs; significantly reduced the arachidonic acid/EPA ratio in plasma and RBCs (suggested by others to be a useful biomarker for arteriosclerotic disease); decreased the concentrations of key fatty acids including arachidonic, palmitic, stearic, oleic and linoleic acids in plasma and arachidonic acid (both doses) and linoleic acid (4 g/day only) in RBCs; increased the proportion of omega-3 fatty acids and decreased the proportion of omega-6 fatty acids in plasma and RBCs; and did not significantly increase the concentration of DHA in plasma or RBCs, indicating that its metabolic effects (including triglyceride lowering) are not due to increases in DHA levels.

Overall, administration of 2 g/day or 4 g/day ultra-pure EPA significantly increased EPA concentrations in a linear, dose-dependent fashion consistent with its triglyceride-lowering effect, caused beneficial shifts in the fatty acid profile, and significantly decreased the arachidonic acid/EPA ratio in plasma and RBCs.

Example 2. Improvement of Cognitive Performance in Subjects with Age-Associated Memory Impairment

A single-center, 6-week, double-blind, randomizes, parallel-group, placebo-controlled, dose-ranging pilot study was performed to evaluate the efficacy, tolerability, and safety of >96% ethyl-EPA in subjects with subjective and objective memory impairment according to generally accepted criteria for Age-Associated Memory Impairment (“AAMI”). The primary objective of the study was to determine the effect of >96% ethyl-EPA 1 g, 2 g, and 4 g daily, compared to placebo, on cognitive performance in subjects with AAMI.

The secondary objectives of this study were the following:

1. To determine the effect of >96% E-EPA on the following tests in the computerized cognitive battery:

-   -   Continuity of attention tasks;     -   Quality of working memory tasks;     -   Quality of episodic memory tasks; and     -   Speed of attention tasks;         2. To determine the safety and tolerability of >96% E-EPA from         routine clinical laboratory tests, adverse events (“AE”)         monitoring, and vital signs; and         3. To determine the potential dose-effect relationship of >96%         E-EPA on the cognitive endpoints by measurement of essential         fatty acids in plasma and red blood cell membranes.

The population for this study was men and women between ages 50 and 70 with self-reported complaints of memory loss, subjective and objective cognitive impairment with a score of at least one standard deviation below that of the mean for age-matched elderly population as determined by the total score of between 13 and 20 from the Paired Associated Learning (“PAL”) subset of the Wechsler Memory Scale, evidence of adequate intellectual function as determined by a scaled score of at least 9 (raw score of at least 32) on the Vocabulary subtest of the Wechsler Adult Intelligence Scale and absence of dementia as determined by a score of 24 or higher on the Mini-Mental State Examination (“MMSE”).

Potential subjects were excluded based on the following exclusion criteria:

-   -   Unlikely or unable to comply with investigational medication         dosing requirements;     -   Diagnosis of major depressive disorder, Alzheimer's or vascular         dementia as defined according to the Mini International         Neuropsychiatric Interview (“MINI”)/Diagnostic and Statistical         Manual of Mental Disorders (4th edition) Text Revision (“TR”)         criteria;     -   Past or current history of:         -   a neurological or psychiatric disorder that could have             affected cognitive function;         -   inflammatory gastrointestinal disease such as Crohn's             Disease or ulcerative colitis;         -   cancer other than basal cell carcinoma;         -   clinically significant cardiac abnormality as measured by             12-lead ECG;     -   Any other medical condition or intercurrent illness not         adequately controlled, which, in the opinion of the study         investigator, may have put the subject at risk when         participating in the study or may have influenced the results of         the study or affected the subject's ability to take part in the         study;     -   Clinically significant abnormal screening results (e.g.,         haematology, biochemistry) on screening or vital signs that fell         outside the normal range for this population, which in the         opinion of the study investigator affected the subject's         suitability for the study;     -   Changes to prescribed medication for a medical condition within         4 weeks of the baseline visit;     -   Omega-3 supplementation within 4 weeks of the baseline visit or         during the study treatment period;     -   Currently taking anticoagulants or daily dose of aspirin greater         than 325 mg.     -   Cough or flu remedies containing opiates or antihistamines         within 2 weeks of the baseline visit or during the 6-week         treatment period; and     -   Known allergy to any ingredients of the study drug or placebo.

Ninety-four subjects were randomized into one of six groups: 1 g E-EPA daily (n=23), 2 g E-EPA daily (n=24), 4 g E-EPA daily (n=24), 1 g placebo daily (n=7), 2 g placebo daily (n=8), and 4 g placebo daily (n=8). E-EPA was provided as 500 mg soft gel capsules containing >96% E-EPA and 0.2% dl-α-tocopherol as an antioxidant. Placebo capsules contained 467 mg of liquid paraffin and 0.2% dl-α-tocopherol. Ninety-one subjects completed the study. Two subjects in the 2 g E-EPA group and one subject in the 2 g placebo group discontinued the study.

The study consisted of a screening visit, a training visit, and four study visits. At the screening visit, subjects' eligibility was determined through cognitive tests (verbal paired associated learning [PAL] subscale, vocabulary subtest, Memory Assessment Clinics Questionnaire [MAC-Q], mini mental state evaluation [MMSE] and MINI [mini international neuropsychiatric interview; sections 1 and 2 of Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) plus dysthymia]), haematology, clinical chemistry and 12-lead electrocardiogram (ECG). At the training visit, subjects were shown how to use the CDR computerized system. Subjects took study drug for 6 weeks and on Days 0, 14, 28 and 42, subjects underwent the CDR cognitive test battery.

At screening cognitive testing and suitability for the study were assessed using the Verbal Paired Associates 1 (Wechsler Memory Scale), Vocabulary Subtest of the WAIS, MAC-Q, MMSE and MINI (DSM-IV Sections 1 and 2 plus Dysthymia).

A selection of tasks from the CDR computerized cognitive assessment system were administered at Visit 2 (training visit), Visit 3 (baseline), Visit 4 (Day 14), Visit 5 (Day 28) and Visit 6 (Day 42). Parallel forms of the tests were presented at each testing session. All tasks were computer-controlled, the information presented on high resolution monitors, and the responses recorded via a response model containing two buttons: one marked ‘no’ and the other ‘yes’. Five CDR composite scores were used as the primary/secondary outcome variables. The task titles were:

-   -   Word Presentation     -   Immediate Word Recall     -   Picture Presentation     -   Simple Reaction Time     -   Digit Vigilance     -   Choice Reaction Time     -   Spatial Working Memory     -   Numeric Working Memory     -   Delayed Word Recall     -   Word Recognition     -   Picture Recognition     -   Bond-Lader Visual Analogue Scales of Mood and Alertness     -   Screen, Using the Computer Mouse

To ensure consistency of approach, full training on the cognitive tests and CDR test battery was provided to study site staff and study subjects. The results of each variable were automatically recorded using the machine interface developed by CDR.

Blood samples (10 mL) were collected at Visit 1 (screening) and at Visits 4, 5 and 6. Analysis was performed by MSR Lipid Analysis, Scottish Crop Research Institute, Dundee, UK. The screening sample acted as baseline for the EFA measurements. Lipid was extracted from plasma, serum and RBC suspensions and converted into fatty acid methyl esters which were analyzed by gas chromatography to give fatty acid profiles as micrograms fatty acid per gram of sample (μgFA/g) and normalized area percent.

All randomized subjects with at least 1 visit post-baseline were included in the Intent to Treat (“ITT”) population, regardless of treatment actually received.

All randomized subjects that completed the study, excluding significant protocol deviators, were defined as the Safety Per Protocol population. An Efficacy Per Protocol population was based on the Efficacy completers. The intercept of the Safety and Efficacy Per Protocol populations defined the Study Per Protocol Population.

All randomized subjects that received at least 1 dose of study medication were included in the Safety Population.

Summary statistics were provided for the ITT and Study Per Protocol Populations separately for all composite scores, major and supportive variables. Summary statistics were performed for both the unadjusted and difference from baseline data (i.e. the difference from the time matched predose assessments on Day 0). Summary statistics were calculated by treatment, day and time-point. The summary statistics comprised n, mean, median, SD, standard error of mean (“SEM”), minimum and maximum values.

Difference from baseline data for each major variable was evaluated by an Analysis of Covariance (“ANCOVA”) using SAS® PROC MIXED Version 8.2. Fixed effects for treatment, day, time point, treatment by day, treatment by time point, treatment by day by time-point were fitted. Subject within treatment was fitted as a repeated effect using the repeated statement. The compound symmetry covariance structure was used. Subjects' time-matched predose assessments on Day 0 were used as a covariate in the analysis.

Least squares means (LS means) were calculated for treatment by day, treatment by time-point and treatment by day by time-point interaction. This formal analysis was conducted for the ITT and Study PP Populations separately.

Safety evaluations were based on the safety population. Safety and tolerability were assessed in terms of AEs, vital signs, 12-lead ECG, clinical laboratory data, medical history, and study drug compliance. Safety and tolerability data were presented by treatment group.

RBC and plasma EFA data were collected at baseline, Day 14, 28 and 42 and summarized by visit for each treatment group. Change from baseline and percent change from baseline were also summarized. ANCOVA comparison of ethyl-EPA dose groups and ethyl-EPA versus placebo was performed.

Efficacy Results.

All CDR cognitive test battery analyses were completed for the ITT and Study PP analysis populations.

For the Intent-to-Treat Analysis for Power of Attention, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. There was no LS mean difference between active treatment and placebo at any time-point.

For the contributing subtasks Simple Reaction Time and Digit Vigilance Speed, there were no statistically significant effects of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. For the subtask measure Choice Reaction Time, there was a statistically significant treatment by day interaction (p=0.011).

For the Study Per-Protocol Power of Attention, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. There was no difference between active treatment and placebo at any time-point.

For the subtasks Simple Reaction Time and Digit Vigilance Speed, there were no statistically significant effects of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. For the subtask measure, Choice Reaction Time, there was a statistically significant treatment by day interaction (p=0.013).

The Intent-to-Treat Continuity of Attention and the contributing subtask Digit Vigilance Targets Detected tests showed no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions.

For the Study Per Protocol Continuity of Attention test, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions.

For the subtask Digit Vigilance Targets Detected, there was a statistically significant treatment by time-point interaction (p=0.040).

For the Intent-to-Treat Quality of Working Memory test, there was a statistically significant treatment by day interaction (p=0.019).

For the contributing subtask Spatial Working Memory Sensitivity Index, there was a statistically significant treatment by day interaction (p=0.015).

For Numeric Working Memory Sensitivity Index, there was a statistical trend for a treatment by day interaction (p=0.089).

For the Study Per-Protocol Quality of Working Memory test, there was a statistically significant treatment by day interaction (p=0.021).

For the contributing subtask Spatial Working Memory Sensitivity Index, there was a statistically significant treatment by day interaction (p=0.014).

For the Intent-to-Treat Quality of Episodic Secondary Memory test, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. The LS mean differences showed overall statistically significant decreases versus placebo for ethyl-EPA 1 g and 2 g (p=0.040 and p=0.035, respectively).

For the contributing subtasks Immediate and Delayed Word Recall Accuracies and for Word and Picture Recognition Sensitivity Indices, there were no statistically significant effects of treatment or treatment by day, treatment by time-point or treatment by day by time-point interactions. For Immediate Word Recall Accuracy, the LS mean differences showed statistically significant decreases for 1 g on Day 14 (p=0.028) and for 2 g on Day 28 (p=0.017). There were statistically significant decreases versus placebo for 1 g and 2 g at AM 1 (p=0.040 and p=0.028, respectively). There were statistically significant decreases for ethyl-EPA 1 g versus placebo on Day 14 at PM 2 (p=0.020) and for 2 g on Day 28 at AM 1 (p=0.006). For Word Recognition Sensitivity Index, the LS mean differences showed statistically significant decreases for ethyl-EPA 1 g on Day 28 (p=0.024) and for 4 g on Day 42 (p=0.038) versus placebo. There was a statistically significant decrease for 4 g at PM 2 (p=0.045) and a statistically significant decrease for 4 g versus placebo on Day 28 at PM 2 (p=0.030). For Picture Recognition Sensitivity Index, the LS mean differences showed statistically significant decrease for 1 g versus placebo on Day 28 at AM 2 (p=0.017) and at PM 2 (p=0.040). For the Study Per-Protocol Quality of Episodic Secondary Memory test, there were no statistically significant effects of treatment, nor treatment by day, treatment by time-point or treatment by day by time-point interactions. The LS mean differences showed overall statistically significant decreases versus placebo for 1 g and 2 g (p=0.043 and p=0.036, respectively).

For the contributing subtasks Immediate and Delayed Word Recall Accuracies and for Word and Picture Recognition Sensitivity Indices, there were no statistically significant effects of treatment or treatment by day, treatment by time-point or treatment by day by time-point interactions. For Immediate Word Recall Accuracy, the LS mean differences to placebo showed statistically significant decreases for ethyl-EPA 1 g on Day 14 (p=0.024) and for 2 g on Day 28 (p=0.017). There were statistically significant decreases for 1 g and 2 g at AM 1 (p=0.038 and p=0.029, respectively) and for 1 g at AM 2 (p=0.048). There were statistically significant decreases for 1 g versus placebo on Day 14 at PM 2 (p=0.019) and for 2 g on Day 28 at AM 1 (p=0.006).

For Word Recognition Sensitivity Index, the LS mean differences to placebo showed statistically significant decreases for 4 g on Day 42 (p=0.038) and for 1 g on Day 28 (p=0.027).

For Picture Recognition Sensitivity Index, the LS mean differences showed statistically significant decreases versus placebo for 1 g on Day 28 at AM 2 (p=0.020) and PM 2 (p=0.026).

For Intent-to-Treat Speed of Memory and the contributing subtasks Spatial and Numeric Working Memory Speeds and Word, and Picture Recognition Speeds, there were no statistically significant effects of treatment, nor treatment by day, treatment by time-point or treatment by day by time-point interactions. For Spatial Working Memory Speed, the LS mean differences showed a statistically significant benefit versus placebo for ethyl-EPA 4 g on Day 14 at PM 1 (p=0.048) and a trend for a benefit for 4 g on Day 42 at AM 1 (p=0.061). For Picture Recognition Speed, there were trends for benefits versus placebo for 1 g on Day 14 at AM 2 (p=0.084) and on Day 28 at AM 1 (p=0.085).

For Study Per-Protocol Speed of Memory and the contributing subtasks Spatial and Numeric Working Memory Speed and Word, and Picture Recognition Speed, there were no statistical significant effects of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions.

For Intent-to-Treat Self-Rated Alertness, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. The LS mean differences showed a statistically significant decrease in ratings for ethyl-EPA 2 g on Day 28 (p=0.047) versus placebo. There was a statistically significant decrease in ratings versus placebo for 2 g on Day 28 at AM 2 (p=0.041). For Study Per-Protocol Self-Rated Alertness, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. The LS mean differences showed a statistically significant decrease in ratings for ethyl-EPA 2 g on Day 28 (p=0.035) versus placebo. There was a statistically significant decrease in ratings versus placebo for 2 g on Day 28 at AM 2 (p=0.033).

For Intent-to-Treat Self-Rated Contentment, there was a statistically significant treatment by day interaction (p<0.001). The LS mean difference to placebo showed no statistically significant effects. For Study Per-Protocol Self-Rated Contentment, there was a statistically significant treatment by day interaction (p<0.001). The LS mean difference to placebo showed no statistically significant effects.

For Intent-to-Treat Self-Rated Calmness, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. For Study Per-Protocol Self-Rated Calmness, there was no statistically significant effect of treatment, nor any treatment by day, treatment by time-point or treatment by day by time-point interactions. The LS mean differences showed a statistical trend for an increase in ratings versus placebo for ethyl-EPA 4 g on Day 42 at PM 1 (p=0.071).

A post-hoc analysis compared the individual placebo groups (1 g, 2 g and 4 g paraffin oil) with the corresponding ethyl-EPA doses.

The pattern of data provided evidence that ethyl-EPA 4 g might improve speed in the attention based measures. For Power of Attention, there was an overall benefit versus the corresponding placebo for 4 g on Day 42. The subtask Simple Reaction Time showed improvements in performance for 4 g at PM 2 collapsed across days and at several time-points on Days 14 and 42. The improvements for 4 g were most pronounced in the Choice Reaction Time task, where there was an overall benefit versus corresponding placebo for 4 g, reflecting a benefit for 4 g over placebo on all study days. The pattern of improvement in performance throughout the assessment days was quite convincing as the improvements began on Day 14 with improvements seen at 2 time points, whereas on Day 42 ethyl-EPA 4 g was superior to placebo at every time point.

For Continuity of Attention, there were isolated declines or improvements in performance, but there was no general pattern of effects and it was considered unlikely these differences were due to the study compound. For Quality of Working Memory and in the subtask measure Numeric Working Memory Sensitivity Index, there were, as in the original analyses, only isolated improvements and declines in performance that were most likely not treatment-related. However, for Spatial Working Memory Sensitivity Index, there was an overall benefit for ethyl-EPA 4 g over placebo on Day 42 in the Study PP Population, which corresponds to the improvements seen for the attention based measures.

For Quality of Episodic Secondary Memory and contributing subtasks, there were a number of decreases for ethyl-EPA that could be explained by the pre-existing differences in performance between the placebo and active treatment groups which was seen in the original analyses. In contrast to the original analysis, the subtask measures of Speed of Memory showed some signs of improvement in performance for active treatment, mostly for 1 g versus placebo. For Self-rated Alertness and Self-rated Contentment, the 1 g dose showed decreases in ratings on Days 14 and 28. However, these decreases were not correlated with a decline in performance in the CDR attention tasks. As with the original planned analysis, there were no differences between active treatment and placebo in Self-Rated Calmness.

Safety Results.

Subjects who used less than 80% of the prescribed dose were to be considered non-compliant; other than those subjects who withdrew for other reasons only 1 subject fell into this category and was withdrawn.

Overall, 139 treatment emergent AEs (“TEAEs”) were reported by 62 (66.0%) of subjects during the study. Most TEAEs were considered mild in severity and unrelated to study drug. More TEAEs were reported for the ethyl-EPA treatment groups (105 events) compared to the placebo treatment groups (34 events). One SAE was reported for the ethyl EPA 2 g treatment group and 3 subjects discontinued due to TEAEs: 2 subjects from the ethyl-EPA 2 g treatment group (the primary reason for discontinuation for 1 of these subjects was non-compliance), and 1 subject from the placebo 2 g treatment group.

There were no deaths during the study. No TEAEs were Definitely Related to the study drug. One subject receiving 1 g ethyl-EPA experienced nausea that was Probably Related to the study drug. Another subject receiving 4 g ethyl-EPA experienced diarrhea that was Probably Related to the study drug; another subject receiving 2 g placebo also experienced diarrhea that was Probably Related to the study drug. Five subjects experienced nausea that was Possibly Related to the study drug; two were in the 1 g ethyl-EPA cohort; one was in the 2 g ethyl-EPA cohort; two were in the 4 g ethyl-EPA cohort. One subject receiving 2 g placebo experienced headache that was Possibly Related to the study drug. All other TEAEs were Not Related or Unlikely Related to the study drug, and included nasopharyngitis (n=3), cystitis (n=2), cough (n=7), toothache (n=2), pharyngolaryngeal pain (n=2), back pain (n=2), pollakiuria (n=2), influenza-like illness (n=2), headache (n=15), diarrhea (n=2), and nausea (n=1).

One subject with a history of transient ischaemic attack, hypertension and osteoarthritis of the hand and osteopaenia receiving 2 g ethyl-EPA experienced worsening epigastric chest pain 17 days after the start of the study and 9 days after the last dose of the study drug. A planned endoscopy revealed oesophagitis and a small hiatus hernia. The subject was treated with omeprazole, which settled her symptoms. The subject had taken felodipine, rosuvastatin, aspirin, glucosamine, and quinine within 14 days of the onset of her symptoms. The study investigator determined that her symptoms were unrelated to the study drug and withdrew the subject from the study. No other Serious Adverse Events occurred during the study.

Essential fatty acid parameters in plasma and RBCs was measured at baseline and on Day 14, 28 and 48 (shown in Tables 7-12).

TABLE 7 EFA Parameter EPA (Plasma and RBC) Mean change from Baseline to Days 14, 28 and 42. EFA Ethyl-EPA Placebo Parameter 1 g 2 g 4 g 1 g 2 g 4 g (μg/g) (N = 23) (N = 24) (N = 24) (N = 7) (N = 8) (N = 8) Plasma Baseline: n    23  24  24  7  8  8 Mean (SD)    48.3 (31.03)  44.9 (25.01)  49.1 (17.23) 47.5 (26.41) 42.1 (16.18) 42.5 (11.86) Day 14: n    23  22  24  7  7  8 Mean (SD)    61.2 (26.61) 124.6 (42.25) 207.7 (57.05)  1.6 (24.69) −1.2 (19.82) 21.9 (32.91) Day 28: n    22  22  24  7  7  8 Mean (SD)    60.3 (36.03) 142.2 (46.23) 215.2 (58.68)  6.5 (15.46)  1.6 (13.64)  1.3 (14.03) Day 42: n    23  22  24  7  7  8 Mean (SD)    62.0 (39.43) 133.4 (43.34) 204.6 (80.69) 11.9 (26.34)  0.4 (21.18)  4.4 (23.32) 1 or 2 g versus 4 g LS Mean −111.8 −60.9 — — — — CI −123.6, −100 −72.7, −49.0 — — — — p-value  <0.001  <0.001 — — — — RBC Baseline: n    23  24  24  7  7  8 Mean (SD)    19.8 (10.85)  18.9 (8.91) 19.8 (5.28) 20.4 (5.77) 19.3 (6.58) 17.2 (4.94) Day 14: n    23  22  24  7  7  8 Mean (SD)    12.3 (7.39)  26.9 (9.15) 39.5 (13.16) −0.5 (6.32)  0.0 (7.17)  2.6 (6.73) Day 28: n    22  22  24  7  7  8 Mean (SD)    14.5 (10.47)  32.9 (10.11) 50.2 (15.82)  1.5 (4.16)  0.0 (7.06)  0.6 (4.42) Day 42: n    23  22  24  7  7  8 Mean (SD)    17.6 (11.89)  38.3 (12.46) 52.5 (20.56) −0.2 (5.90)  1.0 (8.01) −0.2 (6.97) 1 or 2 g versus 4 g LS Mean  −24.4 −11.8 — — — — CI  −27.6, −21.2 −15.0, −8.6 — — — — p-value  <0.001  <0.001 — — — —

TABLE 8 EFA Parameter AA (Plasma and RBC) Mean change from Baseline to Days 14, 28 and 42. EFA Ethyl-EPA Placebo Parameter 1 g 2 g 4 g 1 g 2 g 4 g (μg/g) (N = 23) (N = 24) (N = 24) (N = 7) (N = 8) (N = 8) Plasma Baseline: n  23  24  24  7  8  8 Mean (SD) 202.5 (44.40) 227.3 (42.26) 220.9 (42.80) 210.7 (35.68) 191.6 (28.24) 248.0 (53.52) Day 14: n  23  22  24  7  7  8 Mean (SD)  −9.7 (22.20) −13.9 (22.13) −27.2 (28.89)  0.8 (40.00) −14.4 (19.45)  −5.9 (25.00) Day 28: n  22  22  24  7  7  8 Mean (SD) −11.3 (28.13)  21.6 (28.32) −43.7 (32.24)  3.8 (28.11)  −7.4 (23.72) −16.4 (31.42) Day 42: n  23  22  24  7  7  8 Mean (SD)  −8.7 (31.35) −27.3 (26.76) −48.3 (22.20)  8.2 (20.30) −11.5 (20.88) −11.0 (25.82) 1 or 2 g versus 4 g LS Mean  4.2  15.6 — — — — CI  −8.0, 16.4  3.4, 27.8 — — — — p-value  0.496  0.013 — — — — RBC Baseline: n  23  24  24  7  8  8 Mean (SD) 171.2 (19.79) 172.8 (22.79) 171.0 (25.17) 176.4 (17.65) 152.8 (17.36) 180.4 (23.68) Day 14: n  23  22  24  7  7  8 Mean (SD)  −8.1 (21.95)  −3.1 (25.84) −15.7 (26.76)  −8.5 (22.75)  3.0 (18.20)  −8.1 (27.53) Day 28: n  22  22  24  7  7  8 Mean (SD) −17.0 (20.69) −14.1 (26.89) −22.8 (29.56)  5.2 (22.95)  −2.6 (17.78)  −8.2 (26.89) Day 42: n  23  22  24  7  7  8 Mean (SD) −14.2 (27.69) −18.8 (25.62) −34.4 (31.44)  −9.8 (21.59)  9.7 (16.58) −10.6 (33.49) 1 or 2 g versus 4 g LS Mean  8.4  9.8 — — — — CI  2.0, 14.9  3.3, 16.2 — — — — p-value  0.010  0.003 — — — —

indicates data missing or illegible when filed

TABLE 9 EFA Parameter DHA (Plasma and RBC) Mean change from Baseline to Days 14, 28 and 42. EFA Ethyl-EPA Placebo Parameter 1 g 2 g 4 g 1 g 2 g 4 g (μg/g) (N = 23) (N = 24) (N = 24) (N = 7) (N = 8) (N = 8) Plasma Baseline: n 23 24   24  7  8  8 Mean (SD) 73.1 (30.43) 75.1 (24.02)   78.8 (19.00) 73.7 (14.21) 73.3 (27.74) 76.7 (15.68) Day 14: n 23 22   24  7  7  8 Mean (SD) −6.4 (13.30) −5.4 (14.29) −10.3 (13.35)  0.4 (18.86) −0.8 (14.28) 13.8 (21.05) Day 28: n 22 22   24  7  7  8 Mean (SD) −6.6 (15.53) −8.1 (15.82) −13.5 (14.10)  4.7 (16.31) −0.6 (8.29)  6.0 (17.36) Day 42: n 23 22   24  7  7  8 Mean (SD) −5.4 (18.17) −6.0 (16.69) −13.8 (15.31) 11.8 (21.27)  0.8 (17.57)  6.2 (13.40) 1 or 2 g versus 4 g LS Mean −0.8  1.5 — — — — CI −7.3, 5.7 −5.0, 8.1 — — — — p-value  0.810  0.644 — — — — RBC Baseline: n 23 24   24  7  8  8 Mean (SD) 66.5 (18.65) 64.8 (17.65)   68.3 (14.24) 71.1 (7.48) 66.0 (15.90) 66.2 (15.83) Day 14: n 23 22   24  7  7  8 Mean (SD) −4.6 (9.76) <2.0 (9.46)  −6.9 (9.13) −5.5 (11.93) −0.2 (12.39) −0.4 (12.50) Day 28: n 22 22   24  7  7  8 Mean (SD) −6.4 (11.57) −6.2 (9.34)  −8.7 (11.63)  0.6 (12.86) −0.3 (11.29)  1.1 (12.54) Day 42: n 23 22   24  7  7  8 Mean (SD) −7.0 (12.20) −6.3 (9.42) −13.8 (13.76) −4.1 (12.02)  4.6 (12.94) −0.1 (17.63) 1 or 2 g versus 4 g LS Mean  1.0  1.0 — — — — CI −3.5, 5.4 −3.5, 5.5 — — — — p-value  0.674  0.664 — — — —

TABLE 10 EFA Parameter DPAn-3 (Plasma and RBC) Mean change from Baseline to Days 14, 28 and 42. EFA Ethyl-EPA Placebo Parameter 1 g 2 g 4 g 1 g 2 g 4 g (μg/g) (N = 23) (N = 24) (N = 24) (N = 7) (N = 8) (N = 8) Plasma Baseline: n   23   24 24  7  8  8 Mean (SD)   21.1 (6.62)   19.7 (4.50) 21.7 (4.69) 17.9 (5.18) 18.0 (4.39) 19.0 (2.67) Day14: n   23   22 24  7  7  8 Mean (SD)    7.5 (5.11)   17.4 (7.49) 24.5 (11.28) −0.2 (3.13) −1.0 (3.59)  2.2 (4.98) Day 28: n   22   22 24  7  7  8 Mean (SD)    8.9 (5.62)   19.4 (8.48) 29.7 (13.23)  1.2 (2.06)  0.6 (3.44)  1.3 (3.40) Day 42: n   23   22 24  7  7  8 Mean (SD)   11.3 (6.61)   19.3 (8.63) 32.0 (16.01)  2.2 (3.29)  0.1 (3.61)  0.8 (6.70) 1 or 2 g versus 4 g LS −15.1  −9.5 — — — — CI −17.6, −12.7 −12.0, −7.1 — — — — p-value  <0.001  <0.001 — — — — RBC Baseline: n   23   24 24  7  8  8 Mean (SD)   34.1 (5.43)   33.2 (4.51) 34.5 (4.34) 34.0 (4.27) 33.0 (1.20) 32.4 (2.41) Day 14: n   23   22 24  7  7  8 Mean (SD)    0.9 (5.03)    5.6 (6.28)  5.4 (5.38) −2.8 (4.86) −0.3 (4.96) −0.9 (4.74) Day 28: n   22   22 24  7  7  8 Mean (SD)    3.3 (5.42)    9.4 (6.74) 12.4 (6.98)  0.1 (4.51) −0.8 (4.03) −0.6 (5.19) Day 42: n   23   22 24  7  7  8 Mean (SD)    6.5 (6.19)   13.2 (7.23) 16.2 (10.07) −1.8 (4.64)  2.2 (4.44) −0.9 (6.03) 1 or 2 g versus 4 g LS Mean  −6.2  −2.5 — — — — CI  −7.8, −4,7  −4.1, −1.0 — — — — p-vale  <0.001    0.002 — — — —

TABLE 11 EFA Parameter DGLA (Plasma and RBC) Mean change from Baseline to Days 14, 28 and 42. EFA Ethyl-EPA Placebo Parameter 1 g 2 g 4 g 1 g 2 g 4 g (μg/g) (N = 23) (N = 24) (N = 24) (N = 7) (N = 8) (N = 8) Plasma Baseline: n   23   24   24  7  8  7 Mean (SD)   51.2 (15.01)   53.5 (14.12)   57.1 (14.73) 51.6 (9.20) 41.6 (10.30) 52.6 (7.74) Day 14: n   23   22   24  7  7  8 Mean (SD) −10.4 (10.90) −14.1 (6.88) −22.9 (9.00) −4.1 (8.07) −0.0 (8.63) −1.0 (11.58) Day 28: n   22   22   24  7  7  8 Mean (SD) −10.6 (10.23) −16.2 (9.88) −24.2 (10.73) −4.6 (7.43) −0.6 (5.91)  1.5 (11.78) Day 42: n   23   22   24  7  7  8 Mean (SD)  −9.4 (9.41) −17.3 (9.92) −22.5 (10.87) −3.9 (12.90)  0.9 (9.34)  0.8 (11.04) 1 or 2 versus 4 g LS Mean    3.7    2.5 — — — — CI    0.4, 7.0  −0.9, 5.8 — — — — p-value    0.028    0.143 — — — — RBC Baseline: n   23   24   24  7  8  7 Mean (SD)   23.0 (5.19)   23.0 (5.76)   24.0 (5.77) 22.4 (5.06) 19.7 (5.87) 22.4 (4.91) Day 14: n   23   22   24  7  7  8 Mean (SD)  −2.7 (3.82)  −2.6 (3.54)  −5.3 (4.10) −1.5 (2.08)  0.2 (1.76) −1.8 (4.00) Day 28: n   22   22   24  7  7  8 Mean (SD)  −3.8 (3.31)  −4.5 (3.58)  −7.1 (4.63)  0.2 (3.63) −0.7 (4.06) −0.7 (3.81) Day 42: n   23   22   24  7  7  8 Mean (SD)  −3.5 (4.51)  −5.3 (3.65)  −8.0 (4.98) −1.6 (4.93)  1.9 (3.61) −1.1 (5.31) 1 or 2 versus 4 g LS Mean    1.5    1.5 — — — — CI    0.2, 2.9    1.0, 2.9 — — — — p-value    0.027    0.032 — — — —

TABLE 12 EFA Parameter EPA/AA (Plasma and RBC) Mean change from Baseline to Days 14, 28 and 42. Ethyl-EPA Placebo EFA 1 g 2 g 4 g 1 g 2 g 4 g Parameter (N = 23) (N = 24) (N = 24) (N = 7) (N = 8) (N = 8) Plasma Baseline: n 23 24 24 7   8 8 Mean (SD)  0.2 (0.14)  0.2 (0.12)  0.2 (0.07) 0.2 (0.11)   0.2 (0.10) 0.2 (0.07) Day 14: n 23 22 24 7   7 8 Mean (SD)  0.3 (0.4)  0.6 (0.23)  1.1 (0.28) 0.0 (0.09)   0.0 (0.12) 0.1 (0.12) Day 28: n 22 22 24 7   7 8 Mean (SD)  0.3 (0.20)  0.8 (0.35)  1.3 (0.42) 0.0 (0.08)   0.0 (0.09) 0.0 (0.06) Day 42: n 23 22 24 7   7 8 Mean (SD)  0.3 (0.24)  0.7 (0.29)  1.3 (0.45) 0.0 (0.10)   0.0 (0.12) 0.0 (0.08) 1 or 2 versus 4 g LS Mean −0.66 −0.41 — — — — CI −0.731, −0.475, — — — — −0.597 −0.341 p-value <0.001 <0.001 — — — — RBC Baseline: n 23 24 24 7   8 8 Mean (SD)  0.1 (0.07)  0.1 (0.06)  0.1 (0.04) 0.1 (0.04)   0.1 (0.06) 0.1 (0.03) Day 14: n 23 22 24 7   7 8 Mean (SD)  0.1 (0.04)  0.2 (0.04)  0.3 (0.07) 0.0 (0.03) −0.0 (0.05) 0.0 (0.03) Day 28: n 22 22 24 7   7 8 Mean (SD)  0.1 (0.05)  0.02 (0.06)  0.4 (0.11) 0.0 (0.01) −0.0 (0.04) 0.0 (0.02) Day 42: n 23 22 24 7   7 8 Mean (SD)  0.1 (0.06)  0.3 (0.06)  0.4 (0.14) 0.0 (0.03) −0.0 (0.05) 0.0 (0.03) 1 or 2 versus 4 g LS Mean −0.18 −0.11 — — — — CI −0.204, −0.126, — — — — −0.162  0.085 p-value <0.001 <0.001 — — — —

Notable changes for these parameters occurred in the ethyl-EPA treatment groups at Days 14, 28 and 42 compared to placebo. EPA, DPAn-3 and EPA/AA ratio values increased substantially from baseline, in plasma and RBC, to Day 42 for the ethyl-EPA 1, 2 and 4 g treatment groups, but remained similar to baseline in the placebo treatment groups. AA, DHA and DGLA values decreased substantially from baseline, in plasma and RBC, to day 42 for the ethyl EPA 1, 2 and 4 g treatment groups, but remained similar to baseline in the placebo treatment groups. The difference in EPA, AA (RBC only), DPAn-3, DGLA (1 g only for plasma) and EPA/AA ratio levels in the plasma and RBC were significantly (LS means, p≤0.05) different for the ethyl-EPA 4 g treatment group compared to the ethyl-EPA 1 g and 2 g treatment groups.

Example 3. Ethyl Eicosapentaenoic Acid in Subjects Having Fasting Triglycerides of at Least 500 mg/dL

A multi-center, placebo-controlled randomized, double-blind, 12-week study with an open-label extension is performed to evaluate the efficacy and safety of AMR101 in patients with fasting triglyceride levels ≥500 mg/dL. The primary objective of the study is to determine the efficacy of AMR101 2 g daily and 4 g daily, compared to placebo, in lowering fasting TG levels in patients with fasting TG levels ≥500 mg/dL and ≤1500 mg/dL (≥5.65 mmol/L and 16.94 mmol/L).

The secondary objectives of this study are the following:

-   -   To determine the safety and tolerability of AMR101 2 g daily and         4 g daily;     -   To determine the effect of AMR101 on lipid and apolipoprotein         profiles;     -   To determine the effect of AMR101 on low-density lipoprotein         (LDL) particle number and size;     -   To determine the effect of AMR101 on oxidized LDL;     -   To determine the effect of AMR101 on fasting plasma glucose         (FPG) and hemoglobin A_(1c) (HbA_(1c));     -   To determine the effect of AMR101 on insulin resistance;     -   To determine the effect of AMR101 on high-sensitivity C-reactive         protein (hsCRP);     -   To determine the effects of AMR101 2 g daily and 4 g daily on         the incorporation of fatty acids into red blood cell membranes         and into plasma phospholipids;     -   To explore the relationship between baseline fasting TG levels         and the reduction in fasting TG levels; and     -   To explore the relationship between an increase in red blood         cell membrane eicosapentaenoic acid (EPA) concentrations and the         reduction in fasting TG levels.

The population for this study is men and women (women of childbearing potential will need to be on contraception or practice abstinence) >18 years of age with a body mass index ≤kg/m² who are not on lipid-altering therapy or are currently on lipid-altering therapy. Patients currently on statin therapy (with or without ezetimibe) will be evaluated by the investigator as to whether this therapy can be safely discontinued at screening, or if it should be continued. If statin therapy (with or without ezetimibe) is to be continued, dose(s) must be stable for weeks prior to randomization. Patients taking non-statin, lipid-altering medications (niacin >200 mg/day, fibrates, fish oil, other products containing omega-3 fatty acids, or other herbal products or dietary supplements with potential lipid-altering effects), either alone or in combination with statin therapy (with or without ezetimibe), must be able to safely discontinue non-statin, lipid-altering therapy at screening.

Approximately 240 patients will be randomized at approximately 50 centers in North America, South America, Central America, Europe, India, and South Africa. The study will be a 58- to 60-week, Phase 3, multi-center study consisting of 3 study periods: (1) A 6- to 8-week screening period that includes a diet and lifestyle stabilization and washout period and a TG qualifying period; (2) A 12-week, double-blind, randomized, placebo-controlled treatment period; and (3) A 40-week, open-label, extension period.

During the screening period and double-blind treatment period, all visits are to be within ±3 days of the scheduled time. During the open-label extension period, all visits are to be within ±7 days of the scheduled time. The screening period includes a 4- or 6-week diet and lifestyle stabilization period and washout period followed by a 2-week TG qualifying period. s) must be stable for weeks prior to randomization.

The screening visit (Visit 1) will occur for all patients at either 6 weeks (for patients not on lipid-altering therapy at screening or for patients who will not need to discontinue their current lipid-altering therapy) or 8 weeks (for patients who will require washout of their current lipid-altering therapy at screening) before randomization, as follows:

Patients who do not require a washout: The screening visit will occur at Visit 1 (Week −6). Eligible patients will enter a 4-week diet and lifestyle stabilization period. At the screening visit, all patients will receive counseling regarding the importance of the National Cholesterol Education Program (NCEP) Therapeutic Lifestyle Changes (TLC) diet and will receive instructions on how to follow this diet. Patients who will require a washout: The screening visit will occur at Visit 1 (Week −8). Eligible patients will begin a 6-week washout period at the screening visit. Patients will receive counseling regarding the NCEP TLC diet and will receive instructions on how to follow this diet. Site personnel will contact patients who do not qualify for participation based on screening laboratory test results to instruct them to resume their prior lipid-altering medications.

At the end of the 4-week diet and lifestyle stabilization period or the 6-week diet and stabilization and washout period, eligible patients will enter the 2-week TG qualifying period and will have their fasting TG level measured at Visit 2 (Week −2) and Visit 3 (Week −1). Eligible patients must have an average fasting TG level 500 mg/dL and 1500 mg/dL (5.65 mmol/L and 16.94 mmol/L) to enter the 12-week double-blind treatment period. The TG level for qualification will be based on the average (arithmetic mean) of the Visit 2 (Week −2) and Visit 3 (Week −1) values. If a patient's average TG level from Visit 2 and Visit 3 falls outside the required range for entry into the study, an additional sample for fasting TG measurement can be collected 1 week later at Visit 3.1. If a third sample is collected at Visit 3.1, entry into the study will be based on the average (arithmetic mean) of the values from Visit 3 and Visit 3.1.

After confirmation of qualifying fasting TG values, eligible patients will enter a 12-week, randomized, double-blind treatment period. At Visit 4 (Week 0), patients will be randomly assigned to 1 of the following treatment groups:

-   -   AMR101 2 g daily,     -   AMR101 4 g daily, or     -   Placebo.

During the double-blind treatment period, patients will return to the site at Visit 5 (Week 4), Visit 6 (Week 11), and Visit 7 (Week 12) for efficacy and safety evaluations.

Patients who complete the 12-week double-blind treatment period will be eligible to enter a 40-week, open-label, extension period at Visit 7 (Week 12). All patients will receive open-label AMR101 4 g daily. From Visit 8 (Week 16) until the end of the study, changes to the lipid-altering regimen are permitted (e.g., initiating or raising the dose of statin or adding non-statin, lipid-altering medications to the regimen), as guided by standard practice and prescribing information. After Visit 8 (Week 16), patients will return to the site every 12 weeks until the last visit at Visit 11 (Week 52).

Eligible patients will be randomly assigned at Visit 4 (Week 0) to receive orally AMR101 2 g daily, AMR101 4 g daily, or placebo for the 12-week double-blind treatment period. AMR101 is provided in 1 g liquid-filled, oblong, gelatin capsules. The matching placebo capsule is filled with light liquid paraffin and contains 0 g of AMR101. During the double-blind treatment period, patients will take 2 capsules (AMR101 or matching placebo) in the morning and 2 in the evening for a total of 4 capsules per day. Patients in the AMR101 2 g/day treatment group will receive 1 AMR101 1 g capsule and 1 matching placebo capsule in the morning and in the evening. Patients in the AMR101 4 g/day treatment group will receive 2 AMR101 1 g capsules in the morning and evening.

Patients in the placebo group will receive 2 matching placebo capsules in the morning and evening. During the extension period, patients will receive open-label AMR101 4 g daily. Patients will take 2 AMR101 1 g capsules in the morning and 2 in the evening.

The primary efficacy variable for the double-blind treatment period is percent change in TG from baseline to Week 12 endpoint. The secondary efficacy variables for the double-blind treatment period include the following:

-   -   Percent changes in total cholesterol (TC), high-density         lipoprotein cholesterol (HDL-C), calculated low-density         lipoprotein cholesterol (LDL-C), calculated non-high-density         lipoprotein cholesterol (non-HDL-C), and very low-density         lipoprotein cholesterol (VLDL-C) from baseline to Week 12         endpoint;     -   Percent change in very low-density lipoprotein TG from baseline         to Week 12;     -   Percent changes in apolipoprotein A-I (apo A-I), apolipoprotein         B (apo B), and apo A-I/apo B ratio from baseline to Week 12;     -   Percent changes in lipoprotein(a) from baseline to Week 12         (selected sites only);     -   Percent changes in LDL particle number and size, measured by         nuclear magnetic resonance, from baseline to Week 12 (selected         sites only);     -   Percent change in remnant-like particle cholesterol from         baseline to Week 12 (selected sites only);     -   Percent change in oxidized LDL from baseline to Week 12         (selected sites only);     -   Changes in FPG and HbA_(1c) from baseline to Week 12;     -   Change in insulin resistance, as assessed by the homeostasis         model index insulin resistance, from baseline to Week 12;     -   Percent change in lipoprotein associated phospholipase A2 from         baseline to Week 12 (selected sites only);     -   Change in intracellular adhesion molecule-1 from baseline to         Week 12 (selected sites only);     -   Change in interleukin-6 from baseline to Week 12 (selected sites         only);     -   Change in plasminogen activator inhibitor-1 from baseline to         Week 12 (selected sites only);     -   Change in hsCRP from baseline to Week 12 (selected sites only);     -   Change in serum phospholipid EPA content from baseline to Week         12;     -   Change in red blood cell membrane EPA content from baseline to         Week 12; and     -   Change in serum phospholipid and red blood cell membrane content         in the following fatty acids from baseline to Week 12:         docosapentaenoic acid, docosahexaenoic acid, arachidonic acid,         palmitic acid, stearic acid, and oleic acid.

The efficacy variable for the open-label extension period is percent change in fasting TG from extension baseline to end of treatment. Safety assessments will include adverse events, clinical laboratory measurements (chemistry, hematology, and urinalysis), 12-lead electrocardiograms (ECGs), vital signs, and physical examinations

For TG, TC, HDL-C, calculated LDL-C, calculated non-HDL-C, and VLDL-C, baseline will be defined as the average of Visit 4 (Week 0) and the preceding lipid qualifying visit (either Visit 3 [Week −1] or if it occurs, Visit 3.1) measurements. Baseline for all other efficacy parameters will be the Visit 4 (Week 0) measurement.

For TC, HDL-C, calculated LDL-C, calculated non-HDL-C, and VLDL-C, Week 12 endpoint will be defined as the average of Visit 6 (Week 11) and Visit 7 (Week 12) measurements. Week 12 endpoint for all other efficacy parameters will be the Visit 7 (Week 12) measurement.

The primary efficacy analysis will be performed using a 2-way analysis of covariance (ANCOVA) model with treatment as a factor and baseline TG value as a covariate. The least-squares mean, standard error, and 2-tailed 95% confidence interval for each treatment group and for each comparison will be estimated. The same 2-way ANCOVA model will be used for the analysis of secondary efficacy variables.

The primary analysis will be repeated for the per-protocol population to confirm the robustness of the results for the intent-to-treat population.

The primary efficacy variable will be the percent change in fasting TG levels from baseline to Week 12. A sample size of 69 completed patients per treatment group will provide ≥90% power to detect a difference of 30% between AMR101 and placebo in percent change from baseline in fasting TG levels, assuming a standard deviation of 45% in TG measurements and a significance level of p<0.01. To accommodate a 15% drop-out rate from randomization to completion of the double-blind treatment period, a total of 240 randomized patients is planned (80 patients per treatment group).

Results

Of the 229 randomized subjects, 224 were in the intent-to-treat (“ITT”) population as follows:

-   -   Ultra-pure EPA, 4 g/day: 76 subjects     -   Ultra-pure EPA, 2 g/day: 73 subjects     -   Placebo: 75 subjects

Lipids were extracted from plasma and RBC suspensions and converted into fatty acid methyl esters for analysis using a standard validated gas chromatography/flame ionization detection method. Fatty acid parameters were compared between EPA treatment groups and placebo using an ANCOVA model with treatment, gender, type of statin therapy, and presence of diabetes as factors, and the baseline parameter value as a covariate. LSMs, SEs, and 2-tailed 95% confidence intervals for each treatment group and for each comparison were determined.

Baseline characteristics of the three ITT groups were comparable, with 76% of the ITT subjects being male, 88% being white, having a mean age of 52.9 years, a weight of 95.7 kg and a BMI of 30.8 kg/m². The median triglyceride level of the ITT subjects was 679.5 mg/dl. ITT subjects with incomplete fatty acid data at baseline and/or at 12 weeks were excluded from the analyses described below (as reflected in the n numbers shown in Tables 1-3).

As shown in Table 13, ultra-pure EPA at 2 g/day and 4 g/day both significantly increased mean placebo-adjusted plasma concentrations of EPA, docosapentaenoic acid m-3 (“DPAn-3”), and the ratio of total omega-3 to omega-6 fatty acids (“Ratio of n-3/n-6 FA”). In addition, both 2 g/day and 4 g/day doses of ultra-pure EPA significantly decreased mean placebo-adjusted plasma concentrations of arachidonic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and the plasma ratio of arachidonic acid to EPA. Neither dose of ultra-pure EPA produced a significant change in mean placebo-adjusted plasma concentration of DHA.

TABLE 13 Icosapent ethyl (IPE): change from baseline to week 12 in plasma fatty acid concentrations and ratios. IPE 4 g/day (n = 76) IPE 2 g/day (n = 73) Placebo (n = 75) Mean placebo-adjusted End- End- End- percent change from of- Change of- Change of- Change baseline Base- treat- from Base- treat- from Base- treat- from IPE 4 g/day IPE 2 g/day line ment baseline line ment baseline line ment baseline vs placebo, vs placebo, (SD) (SD) (SE) (SD) (SD) (SE) (SD) (SD) (SE) %, P %, P EPA    50.4   279.8 233.0     50.0   157.5 111.8     41.0  37.4  1.4 792.0 402.3 n = 48, 53, 44 (52.0) (161.3) (15.1) (45.5) (71.0) (14.4) (22.4) (24.3) (15.8) <0.0001 <0.0001 DHA   125.5   118.1 −4.5   118.2   104.8 −13.1     112.2 113.4  −0.7 2.4 −2.3 n = 48, 55, 46 (93.1) (81.6) (6.6) (67.1) (43.5) (6.2) (56.5) (63.6) (6.8) 0.7115 0.7217 DPAn-3    37.5    91.1 54.6    38.0    73.4 36.7    36.7  39.4  4.1 150.8 107.2 n = 46, 56, 47 (20.8) (43.7) (4.3) (19.1) (26.0) (3.9) (13.2) (15.6) (4.3) <0.0001 <0.0001 AA   359.0   280.3 −76.2    368.6   327.3 −36.7     338.7 361.0  17.8 −26.8 −16.4 n = 47, 56, 48 (119.6) (93.9) (11.3) (121.4) (101.4) (10.6) (95.7) (121.4) (11.3) <0.0001 <0.0001 Palmitic 2287 1789 −560    2297 2015 −353   2418 2687   252.1 −34.4 −23.9 n = 44, 54, 48 (1344.1) (1006.9) (138.1) (1070.9) (661.3) (126.9) (1107.2) (1306.6) (135.4) <0.0001 0.0011 Stearic   599.5   500.3 −101      577.9   541.5 −57.8     646.4 709.9  79.6 −28.4 −20.7 n = 47, 55, 47 (317.6) (239.7) (35.4) (229.7) (165.2) (33.3) (306.5) (359.0) (36.0) 0.0002 0.0040 Oleic 2353 1885 −546    2322 2018 −414   2653 2878   274.9 −31.9 −23.8 n = 46, 55, 46 (1499.9) (1130.3) (154.0) (1246.1) (837.7) (142.9) (1460.3) (1616.0) (156.7) 0.0001 0.0023 Linoleic 2098 1882 −309    2113 1981 −227   2374 2521   167.3 −23.7 −15.2 n = 47, 54, 48 (872.3) (910.8) (128.1) (934.8) (775.1) (121.4) (1178.7) (1174.6) (129.0) 0.0003 0.0136 Ratio of    12.3    1.2 −10.8     11.4    2.3 −9.6    11.1  12.2  0.4 −99.4 −88.4 AA/EPA (7.9) (0.9) (0.5) (6.0) (1.4) (0.5) (5.7) (6.0) (0.5) <0.0001 <0.0001 n = 45, 53, 41 Ratio of     0.11     0.24  0.4     0.11     0.17  0.07     0.10   0.10   0.00 145.8 76.8 n-3/n-6 FA (0.050) (0.090) (0.009) (0.062) (0.071) (0.009) (0.036) (0.044) (0.010) <0.0001 <0.0001 n = 49, 55, 46

Mean and standard deviation are reported in Table 13 for baseline and end-of-treatment values; least squares mean and standard error are reported for changes from baseline and placebo-adjusted changes from baseline. Abbreviations for Table 13: AA=arachidonic acid; DHA=docosahexaenoic acid; DPAn-3=docosahexaenoic acid (omega-3); EPA=eicosapentaenoic acid; n-3/n-6 FA=ratio of total omega-3 fatty acids to total omega-6 fatty acids.

As shown in Table 14, ultra-pure EPA at 2 g/day and 4 g/day both significantly increased mean placebo-adjusted concentrations of EPA, DPAn-3, and the ratio of total omega-3 to omega-6 fatty acids (“Ratio of n-3/n-6 FA”) in RBCs. In addition, both 2 g/day and 4 g/day doses of ultra-pure EPA significantly decreased mean placebo-adjusted RBC concentrations of arachidonic acid and the RBC ratio of arachidonic acid to EPA. The RBC concentration of linoleic acid significantly decreased over placebo for subjects in the 4 g/day EPA group. Neither dose of ultra-pure EPA produced a significant change in mean placebo-adjusted RBC concentrations of DHA.

TABLE 14 Icosapent ethyl (IPE): change from baseline to week 12 in red blood cell fatty acid concentrations and ratios. IPE 4 g/day (n = 76) IPE 2 g/day (n = 73) Placebo (n = 75) Mean placebo-adjusted End- End- End- percent change from of- Change of- Change of- Change baseline Base- treat- from Base- treat- from Base- treat- from IPE 4 g/day IPE 2 g/day line ment baseline line ment baseline line ment baseline vs placebo, vs placebo, (SD) (SD) (SE) (SD) (SD) (SE) (SD) (SD) (SE) %, P %, P EPA  11.4  50.2   39.4  10.1  32.8   23.3  10.1  8.2 −1.1 489.6 297.7 n = 50, 56, 45 (6.9) (18.0) (2.0) (5.6) (14.7) (1.9) (5.7) (5.2) (2.1) <0.0001 <0.0001 DHA  62.1  51.7  −9.7  58.3  51.5  −7.3  60.1  54.4 −5.6 −5.2 −1.8 n = 48, 52, 43 (20.0) (16.6) (1.6) (17.1) (14.6) (1.5) (21.3) (19.6) (1.7) 0.1216 0.5859 DPAn-3  28.9  56.9   27.7  28.2  49.8   21.6  29.0  27.0 −2.4 105.6 86.4 n = 48, 53, 40 (5.7) (14.3) (1.5) (6.9) (12.3) (1.4) (4.5) (4.8) (1.6) <0.0001 <0.0001 AA 186.1 148.8 −36.2 179.9 160.3 −20.9 176.0 171.4 −7.6 −15.7 −8.0 n = 47, 53, 44 (34.6) (25.2) (3.4) (34.7) (33.5) (3.2) (27.2) (25.5) (3.5) <0.0001 0.0008 Palmitic 346.1 340.6  −5.8 344.1 341.7  −2.9 341.5 341.2 −1.4 −0.7 −0.1 n = 48, 53, 44 (44.5) (42.6) (5.7) (45.1) (51.9) (5.4) (37.7) (53.3) (6.0) 0.7427 0.9465 Stearic 186.5 178.1  −8.2 181.1 179.0  −4.7 179.8 176.6 −6.5 −0.7 1.1 n = 50, 55, 46 (30.6) (24.8) (3.1) (25.7) (25.0) (2.9) (23.9) (21.4) (3.2) 0.7565 0.5886 Oleic 215.5 208.3  −5.6 216.0 213.0  −1.4 210.3 214.7   4.8 −3.7 −2.1 n = 48, 50, 42 (45.0) (37.1) (4.1) (44.5) (39.5) (4.1) (35.3) (46.3) (4.4) 0.1325 0.3736 Linoleic 188.7 164.7 −25.0 176.3 169.6 −10.7 186.6 187.6 −0.9 −13.0 −4.7 n = 46, 53, 43 (40.6) (40.8) (4.9) (38.5) (42.5) (4.7) (32.4) (42.3) (5.1) <0.0001 0.1278 Ratio of  22.6  3.1 −19.9  22.2  5.2 −17.7  24.1  25.7   2.4 −102 −91.1 AA/EPA (12.4) (1.6) (1.0) (10.5) (2.0) (0.9) (15.4) (13.3) (1.0) <0.0001 <0.0001 n = 48, 54, 44 Ratio of   0.22   0.42    0.20   0.22   0.33    0.12   0.22   0.20  −0.01 106.9 65.5 n-3/n-6 FA (0.072) (0.114) (0.011) (0.068) (0.102) (0.010) (0.076) (0.072) (0.012) <0.0001 <0.0001 n = 50, 56, 43

Mean and standard deviation are reported in Table 14 for baseline and end-of-treatment values; least-squares mean and standard error are reported for changes from baseline and placebo-adjusted changes from baseline. Abbreviations for Table 14: AA=arachidonic acid; DHA=docosahexaenoic acid; DPAn-3=docosahexaenoic acid (omega-3); EPA=eicosapentaenoic acid; n-3/n-6 FA=ratio of total omega-3 fatty acids to total omega-6 fatty acids.

Table 15 shows changes from baseline and placebo-adjusted percent changes from baseline in the proportion (mol %) of fatty acid classes to total fatty acids in both plasma and in RBCs. The monounsaturated fatty acid group included myristoleic, palmitoleic, cis-vaccenic, oleic, gondoic/gadoleic, erucic and nervonic acids. The saturated fatty acid group included myristic, palmitic, stearic, arachidic, behenic and lignoceric acids. The “total omega-3” group included α-linolenic, stearidonic, eicosatrienoic, ω-3 arachidonic, eicosapentaenoic, ω-3 docosapentaenoic and docosahecaenoic acids. The “total omega −6” group included linoleic, γ-linolenic, eicosadienoic, dihomo-γ-linolenic, ω-6 arachidonic, adrenic, and ω-6 docosapentaenoic acids. The “total fatty acid” group represented the sum of the monounsaturated, saturated, total omega-3 and total omega-6 groups. Treatment with 2 g/day or 4 g/day ultra-pure EPA resulted in an increased proportion of omega-3 fatty acids in plasma and in RBCs compared to placebo, and a decreased proportion of omega-6 fatty acids in plasma compared to placebo. Treatment with 2 g/day or 4 g/day ultra-pure EPA also resulted in a decreased proportion of monounsaturated fatty acids in plasma and RBCs compared to placebo. Neither dose induced significant changes in the proportion of saturated fatty acids in plasma or in RBCs.

TABLE 15 Icosapent ethyl: placebo-adjusted percent change in the proportion of fatty acid classes to total^(a) fatty acids. IPE 4 g/day (n = 76) IPE 2 g/day (n = 73) Placebo (n = 75) Mean placebo-adjusted End- End- End- percent change from of- Change of- Change of- Change baseline Base- treat- from Base- treat- from Base- treat- from IPE 4 g/day IPE 2 g/day line ment baseline line ment baseline line ment baseline vs placebo, vs placebo, (SD) (SD) (SE) (SD) (SD) (SE) (SD) (SD) (SE) %, P %, P Plasma Saturated 35.9 34.8 −1.2 36.2 35.8 −0.3 35.6 35.7 −0.2 −2.8 −0.4 n = 49, 56, 49 (3.9) (3.5) (0.5) (3.8) (3.7) (0.5) (3.0) (3.2) (0.5) 0.1049 0.8076 Monounsaturated 30.1 28.7 −1.7 30.7 29.5 −1.4 31.4 32.3   0.9 −7.7 −7.4 n = 49, 56, 49 (5.3) (4.0) (0.5) (5.1) (4.9) (0.5) (4.7) (5.2) (0.5) 0.0008 0.0009 Total omega-6 30.8 29.8 −0.9 30.0 29.8 −0.2 30.0 29.1 −0.9 0.5 3.2 n = 49, 56, 49 (7.2) (6.2) (0.7) (7.1) (6.8) (0.7) (5.8) (5.4) (0.7) 0.8846 0.3044 Total omega-3  3.2  6.7   3.8  3.0  4.8   2.0  2.9  3.0   0.2 132.7 68.4 n = 49, 56, 49 (1.2) (1.9) (0.2) (1.0) (1.3) (0.2) (0.9) (1.1) (0.2) <0.0001 <0.0001 RBCs Saturated 43.5 44.5   0.6 43.8 44.4   0.3 44.2 43.8 −0.3 1.9 1.1 n = 50, 56, 49 (1.4) (2.5) (0.4) (1.8) (3.0) (0.4) (4.2) (2.6) (0.4) 0.1145 0.3278 Monounsaturated 19.6 19.5 −0.2 20.2 20.0 −0.1 20.4 20.8   0.6 −3.9 −3.1 n = 50, 56, 49 (2.2) (1.9) (0.3) (2.8) (1.9) (0.3) (3.0) (2.9) (0.3) 0.0154 0.0435 Total omega-6 30.4 25.5 −4.7 29.8 27.0 −2.9 29.1 29.5 −0.1 −17.3 −13.2 n = 50, 56, 49 (2.2) (2.9) (0.5) (3.2) (3.3) (0.4) (5.1) (3.6) (0.5) <0.0001 0.0014 Total omega-3  6.6 10.4   4.1  6.2  8.7   2.6  6.2  5.9 −0.2 53.4 23.8 n = 50, 56, 49 (1.7) (2.3) (0.3) (1.6) (2.2) (0.3) (2.2) (2.0) (0.3) 0.0008 0.1183

Mean and standard deviation are reported in Table 15 for baseline and end-of-treatment values; least-squares mean and standard error are reported for changes from baseline and placebo-adjusted changes from baseline.

^(a)Total=sum of omega-3, omega-6, monounsaturated, and saturated fatty acids.

The term “Saturated fatty acids” as used in Table 15 refers to the sum of myristic, palmitic, stearic, arachidic, behenic, and lignoceric acids.

The term “Monounsaturated fatty acids” as used in Table 15 refers to myristoleic, palmitoleic, cis-vaccenic, oleic, gondoic/gadoleic, erucic, and nervonic acids.

The term “Omega-6 fatty acids” as used in Table 15 refers to the sum of linoleic, γ-linolenic, eicosadienoic, dihomo-γ-linolenic, omega-6 arachidonic, adrenic, and omega-6 docosapentaenoic acids.

The term “Omega-3 fatty acids” as used in Table 15 refers to the sum of α-linolenic, stearidonic, eicosatrienoic, omega-3 arachidonic, eicosapentaenoic, omega-3 docosapentaenoic, and docosahexaenoic acids.

FIG. 5 shows the placebo-adjusted least squares mean changes (P<0.0001) in EPA concentration from baseline to week 12 in plasma and RBCs. The error bars represent 95% confidence intervals. Treatment with 4 g/day ultra-pure EPA (referred to as “IPE” in the Figures) resulted in a 792.0% placebo-adjusted LS mean increase in plasma EPA over baseline, and a 489.6% placebo-adjusted LS mean increase in RBC EPA over baseline. Treatment with 2 g/day ultra-pure EPA resulted in a 402.3% placebo-adjusted LS mean increase in plasma EPA over baseline, and a 297.7% placebo-adjusted LS mean increase in RBC EPA over baseline.

FIG. 6A shows plasma (dark diamonds) and RBC (light diamonds) placebo-adjusted least square mean changes from baseline to week 12 for select individual fatty acids and ratios of arachidonic acid to EPA (“AA/EPA”) and total omega-3 fatty acids to total omega-6 fatty acids (“n-3/n-6”) upon treatment with 4 g/day ultra-pure EPA. The values for each data point and corresponding P values are reported in Tables 4 and 5. Error bars represent 95% confidence intervals.

FIG. 6B shows plasma (dark diamonds) and RBC (light diamonds) placebo-adjusted least square mean changes from baseline to week 12 for select individual fatty acids and ratios of arachidonic acid to EPA (“AA/EPA”) and total omega-3 fatty acids to total omega-6 fatty acids (“n-3/n-6”) upon treatment with 2 g/day ultra-pure EPA. The values for each data point and corresponding P values are reported in Tables 13 and 14. Error bars represent 95% confidence intervals.

FIG. 7 depicts the ultra-pure EPA dose dependence of changes in EPA plasma and RBC concentration from baseline. Increased doses of ultra-pure EPA resulted in increased EPA concentration in both plasma (dark squares, y-axis on left) and RBCs (light diamonds, y-axis on right). Error bars represent 95% confidence intervals for each data point.

FIGS. 8A and 8B shows the relationship between plasma triglyceride lowering and changes in EPA plasma and RBC concentrations (a pharmacokinetic/pharmacodynamic relationship). Increased EPA concentrations in plasma (FIG. 8A) and RBCs (FIG. 8B) were accompanied by greater reductions in plasma triglyceride levels. Triglyceride concentrations are presented in FIG. 8 as median percent changes from baseline. Vertical error bars represent the first and third quartile of the IQR. EPA concentrations are presented in FIG. 8 as mean percent changes from baseline. Horizontal error bars represent 95% confidence intervals.

These data indicate that ultra-pure EPA at either 2 g/day or 4 g/day significantly increased the concentrations of EPA and its metabolite, DPAn-3, in plasma and RBCs; significantly reduced the arachidonic acid/EPA ratio in plasma and RBCs (suggested by others to be a useful biomarker for arteriosclerotic disease); decreased the concentrations of key fatty acids including arachidonic, palmitic, stearic, oleic and linoleic acids in plasma and arachidonic acid (both doses) and linoleic acid (4 g/day only) in RBCs; increased the proportion of omega-3 fatty acids and decreased the proportion of omega-6 fatty acids in plasma and RBCs; and did not significantly increase the concentration of DHA in plasma or RBCs, indicating that its metabolic effects (including triglyceride lowering) are not due to increases in DHA levels.

Overall, administration of 2 g/day or 4 g/day ultra-pure EPA significantly increased EPA concentrations in a linear, dose-dependent fashion consistent with its triglyceride-lowering effect, increased the concentrations of docosapentaenoic acid n-3 (an EPA metabolite), caused beneficial shifts in the fatty acid profile, and significantly decreased the arachidonic acid/EPA ratio in plasma and RBCs.

Example 4. Pharmacokinetics of Pharmaceutical Compositions Comprising Ethyl

Eicosapentaenoate

A study to characterize the pharmacokinetics of ethyl eicosapentaenoate in plasma and red blood cells after multiple-dose administration of IPE at 2 g per day and 4 g per day was performed in healthy adult male and female humans.

The study was a phase 1, open-label, randomized, multidose study in healthy, nonsmoking men and women aged >18 and ≤55 years with a body mass index (BMI) >18 and ≤30 kg/m². Exclusion criteria included use within 6 weeks prior to randomization through study end of any lipid-altering medications or supplements, including statins, niacin at levels of greater than 200 mg/day, fibrates, ezetimibe, bile acid sequestrants, omega-3 fatty acid medications, and supplements or foods enriched with omega-3 fatty acids (no more than 2 servings of fish per week were permitted).

Following a 14-day screening period, subjects entered a 28-day treatment period and were randomized to one of four IPE dose regimen treatment groups as shown in FIG. 1. Group 1 received IPE 2 g daily (1000-mg capsules; twice-daily [BID] regimen); Group 2 received IPE 4 g daily (1000-mg capsules; BID regimen); Group 3 received IPE 2 g daily (1000-mg capsules; once-daily [QD] regimen); and Group 4 received IPE 2 g daily (500-mg capsules; BID regimen). Treatment was administered orally, with or following a meal in the morning and evening for BID regimens, or in the morning only for the QD regimen. Subjects then entered an 18-day post-treatment pharmacokinetic sampling period.

The safety population was defined as all randomized subjects who received at least one dose of study drug. The per-protocol population was defined as all safety population subjects who completed the 28-day treatment period without any major protocol deviations, and who provided blood samples for EPA analyses.

Assessments and Measurements

Fasting EPA concentrations were measured with a validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method in plasma and RBCs prior to the morning dose (days 1, 14, 26, 28) and at serial time points (1, 3, 5, 6, 8, 10, 12, 24, 48, 72, 120, 192, 312, and 432 hours) after the morning dose on day 28. After the last dose on day 28, EPA concentrations were followed for 18 days to estimate the expected long half-life of EPA. Total plasma EPA included all EPA forms (unesterified EPA and that incorporated in phospholipids, triacylglycerols, and cholesteryl esters). In RBCs, EPA was measured in the cell membrane, where EPA is mainly incorporated in phospholipids.

For total EPA in plasma and RBCs, lipids were isolated by acid/methanol/chloroform extraction followed by centrifugation and purified by isohexane and solid-phase extraction after confirmed complete lipid hydrolysis and transmethylation (acid/methanol, 50° C. overnight). For unesterified EPA in plasma, 25 μL of an inhibitor solution (0.5 g sodium fluoride, 1.0 g L-ascorbic acid, and 0.25 g 5-methylisoxazole-3-carboxylic acid per 10 mL water) was included per 1 mL plasma sample to prevent degradation and lipids were isolated by centrifugation after methanol/chloroform extraction (without hydrolysis or methylation) and further purified by protein precipitation and solid-phase extraction. Quantitation utilized linolenic acid as an internal standard and EPA as the standard for the calibration curve; for total plasma EPA, RBC EPA, and unesterified EPA, the lower limits of quantitation were 10 μg/mL, 5 μg/mL, and 50 ng/mL, respectively. EPA concentrations were measured at Charles River Laboratories Ltd (Elphinstone Research Center, Tranent, Edinburgh, Scotland, UK).

Pharmacokinetic parameters were calculated with standard methods and included area under the plasma concentration versus time curve from time zero to 24 hours (AUC_(0-2hr)), calculated using the linear trapezoidal rule; maximum observed concentration (C_(max)); minimum observed plasma concentration (C_(min), pre-dose, trough concentration); apparent terminal elimination half-life (T_(1/2)) calculated from (log_(e) 2)/λ_(z), where λ_(z) is the apparent terminal rate constant obtained from the slope of the line, fitted by linear least-squares regression, through the terminal points of the natural log of the concentration versus time plots of these points; apparent total plasma clearance (CL/F) after an oral dose, calculated from Dose/AUC₀₋₉₆, where F is the oral bioavailability; time of observed C_(max) (T_(max)); and apparent volume of distribution after an oral dose (V_(z)/F), calculated from Dose/AUC_(0-τ)×λ_(z), where F is the oral bioavailability. Unless otherwise specified, all reported parameters are based on baseline-subtracted concentrations (baseline is due to EPA derived from dietary sources).

The average daily dose of IPE was calculated based on the unused capsule counts across all treatment days prior to the last day of dosing. Percent compliance was calculated as actual daily dose/planned daily dose×100.

Safety assessments consisted of adverse event (AE) monitoring, clinical laboratory measurements (chemistry, hematology, and urinalysis) 12-lead electrocardiographic measurements, vital signs (systolic and diastolic blood pressure, heart rate, respiratory rate, and oral body temperature) and physical examination findings.

Results—Subjects

A total of 48 subjects were randomized to the 4 treatment groups, with 6 male and 6 female subjects in each group. There were 6 subjects who discontinued the study due to withdrawal of consent (n=5) and an AE (n=1; cholecystitis due to previously undiagnosed, longstanding cholelithiasis unrelated to study drug); 42 subjects completed the study (87.5%) (FIG. 1). Mean age (SD) was 38.8 (11.9) years and subjects were primarily white (n=37; 77.1%). Demographic and other baseline characteristics for subjects in the safety population are presented in Table 16:

TABLE 16 Subject Demographics (Safety Population). Group 1 Group 2 Group 3 Group 4 Total Characteristic (n = 12) (n = 12) (n = 12) (n = 12) (n = 48) Age, mean (SD), y 36.9 (13.5) 37.9 (12.9) 39.8 (10.7) 40.7 (11.5) 38.8 (11.9) Male, n (%) 6 (50.0) 6 (50.0) 6 (50.0) 6 (50.0) 24 (50.0) Race, n (%) White 8 (66.7) 10 (83.3) 9 (75.0) 10 (83.3) 37 (77.1) Black 4 (33.3) 2 (16.7) 3 (25.0) 2 (16.7) 11 (22.9) Ethnicity Hispanic 9 (75.0) 9 (75.0) 8 (66.7) 8 (66.7) 34 (70.8) Weight, mean (SD), kg 74.5 (14.1) 75.5 (13.4) 72.9 (11.0) 73.1 (12.1) 74.0 (12.4) BMI, mean (SD), kg/m² 26.2 (2.6) 27.2 (2.6) 26.1 (2.5) 25.7 (3.0) 26.3 (2.6)

Baseline total EPA plasma concentrations were variable between subjects, with a study-wide mean (SD) of 15.3 (15.2) μg/mL. Fourteen subjects had baseline total EPA values lower than the lower limit of quantitation (10 μg/mL). The study-wide mean (SD) baseline unesterified EPA plasma concentration was 0.099 (0.095) μg/mL, which is less than 1% of the total EPA plasma concentration. In RBCs, the study-wide mean (SD) baseline unesterified EPA concentration was 8.01±9.89 μg/mL.

Mean daily IPE dose was 2 g for all groups except Group 2, which had a mean daily IPE dose of 3.9 g. Compliance rates were 99.4%, 97.1%, 99.1%, and 101.3% for Group 1 through 4, respectively.

Pharmacokinetic Results

Following IPE dosing, maximum concentration of total EPA was reached in approximately 5 to 6 hours in plasma (Table 17, FIG. 10) and approximately 8 to 24 hours in RBCs (Table 17). The mean terminal half-life of total EPA in plasma was long, ranging between 70 and 89 hours for the 4 treatment groups. The mean oral clearance and volume of distribution of total EPA in plasma ranged between 684 and 868 mL/hr and 79 and 88 L, respectively (Table 17). Steady state was reached by day 14 in plasma in all treatment groups (FIG. 11). However, RBC concentrations slowly increased over the duration of treatment, and steady state was not reached by day 28 (data not shown). Study-wide steady-state means (SD) for half-life (mainly from plasma lipids), total plasma clearance, and volume of distribution of total EPA were 79 (47) hours, 757 (283) mL/hr, and 82 (56) L, respectively.

TABLE 17 Pharmacokinetic parameters by treatment at day 28 (per-protocol population). Group 1: Group 2: Group 3: Group 4: IPE 2 g/d IPE 4 g/d IPE 2 g/d IPE 2 g/d PK 1 × 1000 mg BID 2 × 1000 mg BID 2 × 1000 mg QD 2 × 500 mg BID Analyte parameter* (n = 10) (n = 9) (n = 12) (n = 12) Total EPA in Baseline (μg/mL) 7.9 (7.0) 19.3 (16.1) 19.2 (17.5) 14.7 (16.6) plasma C_(max) (μg/mL) 154.9 (49.4) 347.2 (112.5) 232.8 (127.6) 210.5 (93.1) AUC_(0-24 hr) (μg · hr/mL) 2907 (1160) 6519 (1963) 2659 (1136) 3233 (1104) C_(min) (μg/mL) 101.0 (50.4) 211.9 (68.2) 75.1 (40.5) 104.2 (42.4) T_(max) (hr) 5 (5, 8) 5 (3, 8) 5 (3, 12) 6 (5, 8) T_(1/2) (hr) 75.1 (46.5) 89.3 (42.0) 69.7 (60.9) 83.5 (38.8) CL/F (mL/hr) 776.4 (256.9) 683.7 (280.6) 867.7 (328.8) 684.5 (248.3) V_(z)/F (L) 79.8 (62.6) 88.4 (55.2) 79.3 (71.5) 79.3 (35.3) Total EPA in Baseline (μg/mL) 5.7 (4.3) 12.1 (15.7) 8.2 (7.1) 6.7 (10.4) RBC C_(max) (μg/mL) 42.3 (14.0) 76.7 (25.2) 31.0 (11.1) 37.6 (15.7) AUC_(0-24 hr) (μg · hr/mL) 801.5 (268.5) 1472 (469.5) 557.6 (239.7) 573.9 (336.4) C_(min) (μg/mL) 31.2 (12.2) 64.1 (21.8) 20.7 (8.6) 26.3 (17.7) T_(max) (hr) 12 (1, 72) 8 (1, 12) 10 (6, 48) 24 (0, 120) T_(1/2) (hr) 683.8 (NC) 371.4 (311.5) 303.2 (NC) 314.4 (194.5) Unesterified Baseline (μg/mL) 0.13 (0.12) 0.08 (0.05) 0.09 (0.06) 0.10 (0.13) EPA in plasma C_(max) (μg/mL) 0.66 (0.34) 1.4 (0.41) 1.4 (0.49) 0.70 (0.24) AUC_(0-24 hr) (μg · hr/mL) 6.9 (3.1) 18.4 (4.6) 9.0 (2.5) 7.5 (2.6) C_(min) (μg/mL) 0.41 (0.28) 1.06 (0.56) 0.36 (0.22) 0.44 (0.24) T_(max) (hr) 5 (0, 48) 5 (0, 24) 5 (3, 8) 3 (0, 24) T_(1/2) (hr) 80.8 (37.5) 97.2 (36.5) 136.3 (80.8) NC CL/F (mL/hr) 364,513 (214,003) 234,329 (81,639) 241,855 (82,203) 326,622 (207,574) V_(z)/F (L) 33,164 (8068) 28,200 (10,929) 40,873 (19,580) NC

A small fraction (≤0.5%) of the total EPA was determined to be unesterified. Based on AUC_(0-24hr) in plasma, 0.3% of the total EPA was unesterified in Group 1 (2 g/day) and Group 2 (4 g/day); based on maximum concentration in plasma, 0.5% and 0.4% of the total EPA was unesterified EPA in group 1 (2 g/day) and group 2 (4 g/day), respectively.

Comparisons of exposure (AUC_(0-24hr) and C_(max)) revealed similarity between QD and BID regimens and between 1×1000-mg and 2×500-mg formulations (FIGS. 4 and 5; Group 1 vs. Group 3, AUC_(0-24hr) for total plasma, total RBCs, and unesterified EPA, with observed differences in maximum concentration expected based on dosing regimen; Group 1 vs. Group 4, AUC_(0-24hr) and maximum concentration for total plasma and unesterified EPA). Dose linearity was observed between IPE 2 g/day and 4 g/day (Group 1 vs. Group 2; dose-normalized comparison of AUC and maximum concentration for total plasma, total RBCs, and unesterified EPA). Based on graphical examination of the AUC_(0-24hr) and maximum concentration of total and unesterified plasma EPA concentrations, no effect of age was observed. Minimum concentration appeared to be slightly lower for males compared with females in some treatment groups, but overall, no consistent gender effect was observed based on all remaining exposure parameters.

Safety and Tolerability

Fourteen (29.2%) subjects reported at least one adverse event (“AE”) during the study (3 [25.0%] in Group 1, 4 [33.3%] in Group 2, 3 [25.0%] in Group 3, and 4 [33.3%] in Group 4). All AEs were treatment emergent and mild or moderate in intensity. The most common AEs were upper respiratory tract infection (two subjects in Group 2) and headache (two subjects in Group 3). One subject in Group 2 discontinued study treatment as noted earlier due to cholecystitis, which was considered unrelated to study drug. There were no clinically meaningful changes in laboratory, electrocardiographic, or physical examination findings.

Discussion

The present study describes the plasma and RBC pharmacokinetics of IPE, a high-purity prescription form of EPA ethyl ester. Once-daily versus twice-daily regimens, 500-mg versus 1000-mg formulations, and 2 g/day versus 4 g/day doses were compared, and the effects of age and gender were also evaluated.

Following oral dosing with IPE 4 g/day and 2 g/day, the study-wide mean (SD) elimination half-life of total EPA at steady state was 79 (47) hours. The study-wide mean apparent total plasma clearance of total EPA at steady state was 757 (283) mL/hr and the apparent volume of distribution of total EPA at steady state was 82 (56) L. Maximum concentrations of total EPA were attained approximately 5 to 6 hours in plasma after dosing. Maximum EPA concentrations occurred later in RBCs compared with plasma (˜8-24 hours after dosing). Steady-state concentrations of total plasma EPA were observed 14 days after continuous dosing, whereas steady state was not reached by day 28 in RBCs.

The slow increase in RBC concentrations observed over the duration of treatment in the present study was consistent with the slow washout of EPA after cessation of dosing (long elimination half-life) in RBCs and likely due to the known slower process of incorporation of EPA into RBC membranes. The maximum plasma concentration of 5 to 6 hours post dosing observed in this study are in agreement with that of a prescription formulation of ethyl EPA available in Japan. The study-wide mean elimination half-life of total plasma EPA of 79 hours was consistent with previously reported mean elimination half-lives ranging from approximately 1 to 3 days for plasma EPA in phospholipids, cholesteryl esters, and triacylglycerol.

Based on dose-normalized maximum observed concentration and AUC of total and unesterified EPA in plasma and total EPA in RBCs, the mean exposure to EPA appears to be dose-proportional between IPE 2 and 4 g/day. This pharmacokinetic dose-linearity was supported by the statistical comparisons of dose-normalized pharmacokinetic values, indicating that IPE has predictable pharmacokinetics. Administration of prescription omega-3-acid ethyl esters (EPA plus DHA) has also been shown to result in dose-dependent increases in serum EPA, but increases in serum DHA were found to be less pronounced and not dose-dependent.

In general, the once-daily and twice-daily dosing regimens for the same daily dose of IPE resulted in similar total AUC for total EPA in plasma. As expected, the maximum observed concentration was higher and the trough concentration was lower in the once-daily regimen compared with the twice-daily regimen. As is typical for drugs with long half-lives, the major determinant of steady-state exposure to EPA in plasma, and even more so in RBCs, was the total daily dose of IPE, irrespective of how the dose was divided over the course of the day. The relatively long half-life of EPA in plasma permits a dosing schedule with intervals of ≥12 hours, and day-to-day variability in the dosing interval is not expected to influence the average plasma or tissue exposure to EPA. In comparing the 500-mg and 1000-mg formulations of IPE at the same daily dose, similar total plasma EPA maximum observed concentrations and AUCs were observed, indicating that, as expected, the 500-mg and 1000-mg formulations provide comparable exposures at comparable doses.

Example 5. Pharmacokinetic and TG-Lowering Pharmacodynamic Effects of Ethyl Eicosapentaenoate Across Clinical Studies

The objective of this Example is to examine the effects of high-purity ethyl eicosapentaenoate (“IPE”) on EPA concentrations in plasma and RBCs in response to dose and the relationship to triglyceride (“TG”) lowering across 3 clinical studies.

MARINE and ANCHOR were 12-week, phase 3, double-blind studies that randomized patients to IPE 4 g/day, 2 g/day, or placebo. MARINE randomized 229 patients with TG≥500 and ≤2000 mg/dL while ANCHOR randomized 702 high-risk patients with TG≥200 and <500 mg/dL despite LDL-C control while on statin therapy. IPE 4 g/day and 2 g/day were also investigated in 48 healthy subjects for 4 weeks in a phase 1 pharmacokinetic (“PK”) study.

In all studies, a greater increase in EPA concentrations in both plasma and RBCs was observed with IPE 4 g/day than with 2 g/day, indicating a dose-dependent increase in EPA exposure. EPA concentration data from healthy volunteers and in patients with hypertriglyceridemia indicate that the PK of EPA are linear with dose. In MARINE and ANCHOR, median percent reductions in TG from baseline were higher with IPE 4 g/day than with 2 g/day and demonstrated a dose-proportional relationship. In plasma and RBCs, a linear PD relationship between EPA levels and TG reduction was also observed, demonstrating a linear concentration-response relationship.

Taken together, the linear-dose-PK and dose-concentration-response PD relationships indicate that the PK/PD of IPE are predictable, as the results demonstrate a trend of increasing TG-lowering efficacy with respect to both the IPE dose and EPA concentration in plasma and RBCs.

A phase 1 PK study conducted in healthy volunteers demonstrated that the PK profile of IPE was that of a slowly cleared and extensively distributed molecule with dose-linearity.

The MARINE and ANCHOR pivotal studies demonstrated collectively that IPE significantly reduced TG levels in patients with very high (MARINE) or high (ANCHOR) TG levels and on statin therapy without raising LDL-C.

The present analysis evaluated data from 2 studies conducted in patients with elevated TG levels and 1 study conducted in healthy volunteers for a PK/PD dose-response relationship with respect to IPE dose, EPA concentration in plasma and RBCs, and reduction in TG levels.

Study Designs:

-   -   Phase 1 PK study         -   Open-label, randomized, multidose study with a 14-day             screening period followed by a 4-week treatment period         -   Healthy, nonsmoking volunteers aged >18 and ≤55 years not             receiving any lipid-altering medications or supplements             within 6 weeks before the study were randomized to 4 IPE             dose regimens (groups 1 and 2 are included in this             analysis):             -   Group 1: IPE 2 g/day (one 1000-mg capsule BID)             -   Group 2: IPE 4 g/day (two 1000-mg capsules BID)             -   Group 3: IPE 2 g/day (two 1000-mg capsules QD)             -   Group 4: 2 g/day (two 500-mg capsules BID)         -   After dosing, subjects entered an 18-day post-treatment PK             sampling period     -   MARINE and ANCHOR1,2         -   Phase 3, placebo-controlled, randomized, double-blind,             multicenter studies with a 4- to 6-week lead-in period of             diet, lifestyle, and medication stabilization with washout             of prohibited lipid-altering medications         -   In both studies, patients aged >18 years with qualifying             lipid levels (MARINE: TG≥500 mg/dL and ≤2000 mg/dL; ANCHOR:             TG≥200 and <500 mg/dL and LDL-C≥40 and ≤115 mg/dL) entered a             12-week, double-blind treatment period and were randomized             to receive either IPE 4 g/day, IPE 2 g/day, or matched             placebo         -   In the MARINE study, stable statin therapy with or without             ezetimibe was permitted but not required         -   In the ANCHOR study, patients were required to be at high             risk for cardiovascular disease as defined by the NCEP ATP             III guidelines3 and on stable statin dose (atorvastatin,             rosuvastatin, or simvastatin with or without ezetimibe)

Assessments and Measurements

In the MARINE and ANCHOR studies, the trough EPA concentrations were measured after 12 weeks of dosing with placebo, 2 g/day or 4 g/day IPE

In the Phase 1 PK study, the trough EPA concentrations were measured after 28 days of dosing with 2 or 4 g/day IPE (there was no placebo group; a value of zero is used in plots as a point of reference)

EPA concentrations were measured with a validated LC-MS/MS method in plasma and in RBCs in all 3 studies

Total plasma EPA included all EPA forms (unesterified EPA and that incorporated in phospholipids, triacylglycerols, and cholesteryl esters); in RBCs, EPA was from cell membranes, where it is mainly incorporated in phospholipids

Total EPA in plasma and RBCs: lipids were isolated by acid/methanol/chloroform extraction followed by centrifugation and purified by isohexane and solid-phase extraction after confirmed complete lipid hydrolysis and transmethylation (acid/methanol, 50° C. overnight)

Quantitation utilized linolenic acid as an internal standard and EPA as the standard for the calibration curve; for total plasma EPA and total RBC EPA, the lower limits of quantitation were 10 μg/mL and 5 μg/mL, respectively

TG levels were evaluated as in the MARINE and ANCHOR studies as previously reported.

Serum TG levels were measured with enzymatic colorimetric tests with calibration directly traceable to US CDC reference procedures

Results

Subjects

Baseline characteristics are summarized in Table 18 and were comparable among treatment groups within each study.

TABLE 18 Demographics and Baseline Characteristics. Phase 1 PK Study MARINE ANCHOR IPE IPE IPE IPE IPE IPE 4 g/day 2 g/day 4 g/day 2 g/day Placebo 4 g/day 2 g/day Placebo Characteristic (n = 12) (n = 12) (n = 77) (n = 76) (n = 76) (n = 223) (n = 236) (n = 233) Age, mean 37.9 36.9 51.9 53.4 53.4 61.1 61.8 61.2 (SD), y (12.9) (13.5) (10.3) (9.3) (8.3) (10.0) (9.4) (10.1) Male, n (%)  6    6   59   58   58   142   144   145   (50.0) (50.0) (76.6) (76.3) (76.3) (60.9) (61.0) (62.2) Weight, mean 75.5 74.5 93.2 92.1 93.0 94.5 95.5 97.0 (SD), kg (13.4) (14.1) (18.3) (15.6) (16.9) (18.3) (18.3) (19.1) BMI, mean 27.2 26.2 30.4 30.8 31.0 32.7 32.9 33.0 (SD), kg/m² (2.6) (2.6) (4.3) (4.2) (4.3) (5.0) (5.0) (5.0) Diabetes, n NA NA 22   20   21   171   172   171   (%) (28.6) (26.3) (27.6) (73.4) (72.9) (73.4) TG, median NA NA 679.5  656.5  703.0  264.8  254.0  259.0  (IQR), mg/dL (265.3) (303.5) (426.5) (93.0) (92.5) (81.0) n = 76 n = 73 n = 75 n = 226 n = 234 n = 227 Total plasma 19.3  7.9 61.2 63.6 57.7 28.1 28.1 28.1 EPA, mean (16.1) (7.0) (67.4) (51.4) (42.7) (18.8) (13.7) (28.0) (SD), ug/mL n = 9 n = 10 n = 69 n = 62 n = 61 n = 71 n = 73 n = 81 RBC EPA, 12.1  5.7 16.0 15.7 14.7 11.6 10.9 11.2 mean (SD), (15.7) (4.3) (9.2) (9.9) (9.3) (5.6) (5.2) (6.6) ug/mL n = 9 n = 10 n = 66 n = 61 n = 64 n = 69 n = 71 n = 79

EPA Dose Response

Both healthy subjects and patients with hypertriglyceridemia experienced dose-dependent increases in plasma EPA concentrations following treatment with IPE (FIG. 15).

Increasing doses of IPE led to greater increases in EPA concentrations in both plasma and RBCs in all three studies, as shown in FIGS. 16 and 17. Mean concentrations shown in FIGS. 16-17 are based on baseline-subtracted trough concentrations of total EPA (change from baseline) at steady state.

As shown in FIGS. 18 and 19, subjects in the MARINE and ANCHOR studies treated with IPE experienced dose-dependent reduction in triglyceride levels that also was linearly associated with an increase in EPA levels as measured in both plasma (FIGS. 18A, 19A) and red blood cells (FIGS. 18B, 19B).

Data from both the MARINE and ANCHOR studies demonstrate that gender, age, baseline TG levels, body weight, and BMI have no effect on EPA concentrations in plasma and RBCs.

Treatment with concomitant medications including statins, antihypertensive medications, and antiplatelet medications did not affect increases in plasma EPA concentrations in both the MARINE and ANCHOR studies.

Analysis of patients in ANCHOR indicated that patients with and without diabetes mellitus had on average the same plasma and RBC EPA increases.

IPE was administered with or following a meal (or snack) and therefore exposure measurements of EPA were studied in the fed state.

Plasma and RBC EPA levels were increased compared with baseline in both the 2 and 4 g/day groups in all 3 studies, and were linear with dose, indicating that IPE has predictable pharmacokinetics.

The relationships between IPE dose and TG-lowering response and between EPA concentration and TG-lowering response were similar and linear, indicating that IPE has predictable PD and PK/PD characteristics.

Demographic factors (age, gender, body weight, BMI, and presence of diabetes mellitus), baseline TG levels, and concomitant medications (statins, antihypertensives, and antiplatelets) did not affect the increase in EPA concentrations in plasma and RBCs after treatment with IPE.

The PK results were similar among all three studies; the PK/PD results were similar between the MARINE and ANCHOR studies. 

1-87. (canceled)
 88. A method of reducing triglycerides in a subject having a fasting baseline triglyceride level of at least about 700 mg/dl, a body mass index (BMI) of at least about 31 kg/m², and on a statin therapy, the method comprising administering to the subject about 4 g of ethyl eicosapentaenoate per day.
 89. The method of claim 88, wherein the ethyl eicosapentaenoate is present in a pharmaceutical composition and the ethyl eicosapentaenoate comprises at least about 96 wt. % of all omega-3 fatty acids in the pharmaceutical composition.
 90. The method of claim 89, wherein about 1 g of the pharmaceutical composition is present in each of 4 capsules.
 91. The method of claim 88, wherein the subject has a baseline LDL-C level of about 40 mg/dL to about 100 mg/dL.
 92. The method of claim 88, wherein the subject is diabetic.
 93. The method of claim 88, wherein the subject exhibits at least about a 10% reduction in fasting triglycerides compared to a baseline triglyceride level.
 94. The method of claim 93, wherein the subject exhibits no statistically significant increase in LDL-C compared to a LDL-C level prior to initial administration of the ethyl eicosapentaenoate.
 95. The method of claim 88, wherein the statin therapy is a stable statin therapy.
 96. The method of claim 88, wherein the subject is administered the ethyl eicosapentaenoate for at least about 12 weeks.
 97. The method of claim 88, further comprising reducing a risk of stroke, myocardial infarction, or both in the subject.
 98. A method of reducing triglycerides in a subject having a fasting baseline triglyceride level of at least about 500 mg/dl, a body mass index (BMI) of at least about 31 kg/m², and on a statin therapy, the method comprising administering to the subject about 4 g of ethyl eicosapentaenoate per day.
 99. The method of claim 98, wherein the ethyl eicosapentaenoate is present in a pharmaceutical composition and the ethyl eicosapentaenoate comprises at least about 96 wt. % of all omega-3 fatty acids in the pharmaceutical composition.
 100. The method of claim 99, wherein about 1 g of the pharmaceutical composition is present in each of 4 capsules.
 101. The method of claim 98, wherein the subject has a baseline LDL-C level of about 40 mg/dL to about 100 mg/dL.
 102. The method of claim 98, wherein the subject is diabetic.
 103. The method of claim 98, wherein the subject exhibits at least about a 10% reduction in fasting triglycerides compared to a baseline triglyceride level.
 104. The method of claim 103, wherein the subject exhibits no statistically significant increase in LDL-C compared to a LDL-C level prior to initial administration of the ethyl eicosapentaenoate.
 105. The method of claim 98, wherein the statin therapy is a stable statin therapy.
 106. The method of claim 98, wherein the subject is administered the ethyl eicosapentaenoate for at least about 12 weeks.
 107. The method of claim 98, further comprising reducing a risk of stroke, myocardial infarction, or both in the subject. 